Composition for collecting and preserving placental stem cells and methods of using the composition

ABSTRACT

The present invention provides improved compositions and methods for the collection of stem cells from an organ, e.g., placenta. The invention provides a stem cell collection composition comprising an apoptosis inhibitor and, optionally, an enzyme such as a protease or mucolytic enzyme, vasodilator, necrosis inhibitor, oxygen-carrying perfluorocarbon, or an organ preserving compound. The invention provides methods of using the stem cell collection composition to collect stem cells and to preserve populations of stem cells.

This application claims benefit of U.S. Provisional Application No.60/754,969, filed Dec. 29, 2005, the entirety of which is incorporatedherein by reference.

1. INTRODUCTION

The present invention relates to improved methods and compositions forthe collection of placental stem cells, e.g., by perfusion or physicaland/or enzymatic disruption of a placenta or a part thereof, and methodsof collecting the stem cells using the compositions. The compositionsdisclosed herein comprise a physiologically-acceptable aqueous solution,e.g., a saline solution, one or more proteases and one or moreinhibitors of JNK (c-Jun-N-terminal Kinase). Optionally, the compositionfurther comprises a compound that modulates (e.g., inhibits) TNF-α, animmunomodulatory compound, a vasodilator, necrosis inhibitor,oxygen-carrying perfluorocarbon, or a combination of any of theforegoing. The present invention provides methods of using thecomposition to collect and preserve stem cells and populations of stemcells.

2. BACKGROUND OF THE INVENTION

Human stem cells are totipotential or pluripotential precursor cellscapable of generating a variety of mature human cell lineages. Evidenceexists that demonstrates that stem cells can be employed to repopulatemany, if not all, tissues and restore physiologic and anatomicfunctionality.

Many different types of mammalian stem cells have been characterized.See, e.g., Caplan et al., U.S. Pat. No. 5,486,359 (human mesenchymalstem cells); Hu et al., WO 00/73421 (methods of isolation,cryopreservation, and therapeutic use of human amniotic epithelialcells); Beltrami et al., Cell 114(6):763-766 (2003) (cardiac stemcells); Forbes et al., J. Pathol. 197(4):510-518 (2002) (hepatic stemcells). Umbilical cord blood, and total nucleated cells derived fromcord blood, have been used in transplants to restore, partially orfully, hematopoietic function in patients who have undergone ablativetherapy.

The placenta is a particularly attractive source of stem cells. See,e.g., Hariri, U.S. Patent Application Publication Nos. 2002/0123141 and2003/0032179. While placentas are readily available, it is desirable tomaximize the number of stem cells obtained from each placenta. Stemcells, like other types of cells, are sensitive to environmental changesbrought about during collection and storage. These changes can bringabout apoptosis or necrosis of the stem cells. There is thus a need forimproved compositions and for the collection of placental stem cellsfrom a post-partum mammalian placenta so as to recover increased numbersof stem cells from a single placenta.

3. SUMMARY OF THE INVENTION

The present invention provides improved methods and compositions for theperfusion and preservation of organs, e.g., placenta, to facilitate therecovery of stem cells, as well as methods of collecting stem cellsusing the compositions.

In one embodiment, the invention provides a method of isolating a stemcell, comprising contacting said stem cell with a solution comprising aninhibitor of apoptosis, and isolating said stem cell. In anotherembodiment, the invention provides a method of isolating a stem cell,comprising contacting the stem cell with a solution comprising anecrosis inhibitor, and isolating said stem cell.

In a specific embodiment of either of the above embodiments, saidinhibitor of apoptosis is a caspase inhibitor. In another specificembodiment, said inhibitor of apoptosis is a c-Jun N-terminal kinase(JNK) inhibitor. In a more specific embodiment, said JNK inhibitor doesnot modulate the differentiation or proliferation of said stem cellprior to said isolation. In another specific embodiment, the methodadditionally comprises contacting said stem cell with a compound thatinhibits necrosis. In another specific embodiment, said stem cell isisolated from mammalian placenta, umbilical cord, placental blood orumbilical cord blood. In another specific embodiment, the methodadditionally comprises contacting said stem cell with an oxygen-carryingperfluorocarbon. In another specific embodiment, the method additionallycomprises contacting said stem cell with a protease. In a more specificembodiment, said protease is a matrix metalloprotease or a neutralprotease. In a more specific embodiment, said matrix metalloprotease iscollagenase. In another specific embodiment, said neutral protease isthermolysin or dispase. In another specific embodiment, the methodadditionally comprises contacting said stem cell with a mucolyticenzyme. In a more specific embodiment, said mucolytic enzyme ishyaluronidase. In another specific embodiment, of the method, saidsolution is a saline solution or culture medium. In another specificembodiment, said solution additionally comprises hydroxyethyl starch,lactobionic anion and raffinose. In another specific embodiment, saidsolution comprises UW solution.

In another specific embodiment, said stem cell is isolated from a tissueby physical disruption, including enzymatic digestion, of said tissue.In a more specific embodiment, said stem cell is isolated from theplacenta by enzymatic digestion of at least a part of the placenta.

In another specific embodiment, said mammalian placenta is exsanguinatedprior to said physical disruption. In another specific embodiment, saidstem cell is isolated from said mammalian placenta, and said isolatingis performed by perfusing said mammalian placenta with a perfusionsolution. In a more specific embodiment, said perfusion is performed byperfusing the mammalian placenta with said perfusion solution in anamount and for a time sufficient to collect a detectable number of stemcells from said mammalian placenta. In more specific embodiment, saidmammalian placenta is exsanguinated prior to said perfusing. In morespecific embodiment, said perfusing is performed by passing saidperfusion solution into one or both of the umbilical artery andumbilical vein of said placenta. In another more specific embodiment,said perfusion solution comprises 0.9% NaCl solution or phosphatebuffered saline. In another more specific embodiment, said perfusinguses from about 500 mL to about 2000 mL of said perfusion solution, orabout 750 mL of said perfusion solution. In another more specificembodiment, said perfusing is performed a plurality of times.

In another specific embodiment, said stem cell is exposed to a hypoxiccondition for less than 72 hours following delivery, wherein a hypoxiccondition is a concentration of oxygen that is less than normal bloodoxygen concentration. In a more specific embodiment, said stem cell isexposed to said hypoxic condition for less than 48 hours. In anothermore specific embodiment, said stem cell is exposed to said hypoxiccondition for less than 24 hours. In another more specific embodiment,said stem cell is exposed to said hypoxic condition for less than sixhours. In another more specific embodiment, said stem cell is notexposed to a hypoxic condition.

In another specific embodiment, said stem cell is not exposed to shearstress during said isolation.

In another specific embodiment, said JNK inhibitor is an indazole. Inanother specific embodiment, said JNK inhibitor has the structure

wherein:

A is a direct bond, —(CH₂)_(a)—, —(CH₂)_(b)CH═CH(CH₂)_(c)—, or—(CH₂)_(b)C≡C(CH₂)_(c)—;

R₁ is aryl, heteroaryl or heterocycle fused to phenyl, each beingoptionally substituted with one to four substituents independentlyselected from R₃;

R₂ is —R₃, —R₄, —(CH₂)_(b)C(═O)R₅, —(CH₂)_(b)C(═O)OR₅,—(CH₂)_(b)C(═O)NR₅R₆,

—(CH₂)_(b)C(═O)NR₅(CH₂)_(c)C(═O)R₆, —(CH₂)_(b)NR₅C(═O)R₆,

—(CH₂)_(b)NR₅C(═O)NR₆R₇, —(CH₂)_(b)NR₅R₆, —(CH₂)_(b)OR₅,

—(CH₂)_(b)SO_(d)R₅ or —(CH₂)_(b)SO₂NR₅R₆;

a is 1, 2, 3, 4, 5 or 6;

b and c are the same or different and at each occurrence independentlyselected from 0, 1, 2, 3 or 4;

d is at each occurrence 0, 1 or 2;

R₃ is at each occurrence independently halogen, hydroxy, carboxy, alkyl,alkoxy, haloalkyl, acyloxy, thioalkyl, sulfinylalkyl, sulfonylalkyl,hydroxyalkyl, aryl, arylalkyl, heterocycle, heterocycloalkyl, —C(═O)OR₈,—OC(═O)R₉, —C(═O)NR₈R₉, —C(═O)NR₈OR₉, —SO₂NR₈R₉, —NR₈SO₂R₉, —CN, —NO₂,—NR₈R₉, —NR₈C(═O)R₉, —NR₈C(═O)(CH₂)_(b)OR₉, —NR₈C(═O)(CH₂)_(b)R₉,—O(CH₂)_(b)NR₈R₉, or heterocycle fused to phenyl;

R₄ is alkyl, aryl, arylalkyl, heterocycle or heterocycloalkyl, eachbeing optionally substituted with one to four substituents independentlyselected from R₃, or R₄ is halogen or hydroxy;

R₅, R₆ and R₇ are the same or different and at each occurrenceindependently hydrogen, alkyl, aryl, arylalkyl, heterocycle orheterocycloalkyl, wherein each of R₅, R₆ and

R₇ are optionally substituted with one to four substituentsindependently selected from R₃; and

R₈ and R₉ are the same or different and at each occurrence independentlyhydrogen, alkyl, aryl, arylalkyl, heterocycle, or heterocycloalkyl, orR₈ and R₉ taken together with the atom or atoms to which they are bondedform a heterocycle, wherein each of R₈, R₉, and R₈ and R₉ taken togetherto form a heterocycle are optionally substituted with one to foursubstituents independently selected from R₃.

In another specific embodiment, said JNK inhibitor has the structure

wherein:

R₁ is aryl or heteroaryl optionally substituted with one to foursubstituents independently selected from R₇;

R₂ is hydrogen;

R₃ is hydrogen or lower alkyl;

R₄ represents one to four optional substituents, wherein eachsubstituent is the same or different and independently selected fromhalogen, hydroxy, lower alkyl and lower alkoxy;

R₅ and R₆ are the same or different and independently —R₈,—(CH₂)_(a)C(═O)R₉, —(CH₂)_(a)C(═O)OR₉, —(CH₂)_(a)C(═O)NR₉R₁₀,—(CH₂)_(a)C(═O)NR₉(CH₂)_(b)C(═O)R₁₀, —(CH₂)_(a)NR₉C(═O)R₁₀,(CH₂)_(a)NR₁₁C(═O)NR₉R₁₀, —(CH₂)_(a)NR₉R₁₀, (CH₂)_(a)OR₉,—(CH₂)_(a)SO_(c)R₉ or —(CH₂)_(a)SO₂NR₉R₁₀;

or R₅ and R₆ taken together with the nitrogen atom to which they areattached to form a heterocycle or substituted heterocycle;

R₇ is at each occurrence independently halogen, hydroxy, cyano, nitro,carboxy, alkyl, alkoxy, haloalkyl, acyloxy, thioalkyl, sulfinylalkyl,sulfonylalkyl, hydroxyalkyl, aryl, arylalkyl, heterocycle, substitutedheterocycle, heterocycloalkyl, —C(═O)OR₈, —OC(═O)R₈, —C(═O)NR₈R₉,—C(═O)NR₈OR₉, —SO_(c)R₈, —SO_(c)NR₈R₉, —NR₈SO_(c)R₉, —NR₈R₉,—NR₈C(═O)R₉, —NR₈C(═O)(CH₂)_(b)OR₉, —NR₈C(═O)(CH₂)_(b)R₉,—O(CH₂)_(b)NR₈R₉, or heterocycle fused to phenyl;

R₈, R₉, R₁₀ and R₁₁ are the same or different and at each occurrenceindependently hydrogen, alkyl, aryl, arylalkyl, heterocycle,heterocycloalkyl;

or R₈ and R₉ taken together with the atom or atoms to which they areattached to form a heterocycle;

a and b are the same or different and at each occurrence independentlyselected from 0, 1, 2, 3 or 4; and

c is at each occurrence 0, 1 or 2.

In another specific embodiment, said JNK inhibitor has the structure

wherein R₀ is —O—, —S—, —S(O)—, —S(O)₂—, NH or —CH₂—;

the compound of structure (III) being: (i) unsubstituted, (ii)monosubstituted and having a first substituent, or (iii) disubstitutedand having a first substituent and a second substituent;

the first or second substituent, when present, is at the 3, 4, 5, 7, 8,9, or 10 position, wherein the first and second substituent, whenpresent, are independently alkyl, hydroxy, halogen, nitro,trifluoromethyl, sulfonyl, carboxyl, alkoxycarbonyl, alkoxy, aryl,aryloxy, arylalkyloxy, arylalkyl, cycloalkylalkyloxy, cycloalkyloxy,alkoxyalkyl, alkoxyalkoxy, aminoalkoxy, mono-alkylaminoalkoxy,di-alkylaminoalkoxy, or a group represented by structure (a), (b), (c),(d), (e), or (f):

wherein R₃ and R₄ are taken together and represent alkylidene or aheteroatom-containing cyclic alkylidene or R₃ and R₄ are independentlyhydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,aryloxyalkyl, alkoxyalkyl, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonylalkyl, amino, mono-alkylamino,di-alkylamino, arylamino, arylalkylamino, cycloalkylamino,cycloalkylalkylamino, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl.

In a more specific embodiment, the method additionally comprisescontacting the stem cell with an immunomodulatory compound. In a morespecific embodiment, said immunomodulatory compound is3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione;3-(4′aminoisolindoline-1′-onw)-1-piperidine-2,6-dione;4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; orα-(3-aminophthalimido) glutarimide. In another more specific embodiment,said immunomodulatory compound is a compound having the structure

wherein one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl, or a pharmaceutically acceptable salt,hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, ormixture of stereoisomers thereof. In another more specific embodiment,said immunomodulatory compound is a compound having the structure

wherein one of X and Y is C═O and the other is CH₂ or C═O;

R¹ is H, (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴,(C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³,C(S)NHR³, C(O)NR³R^(3′), C(S)NR³R³ or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl;

R³ and R^(3′) are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl,(C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, or(C₀-C₄)alkyl-(C₂-C₅)heteroaryl;

R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, or(C₂-C₅)heteroaryl;

each occurrence of R⁶ is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₂-C₅)heteroaryl, or(C₀-C₈)alkyl-C(O)O—R⁵ or the R⁶ groups can join to form aheterocycloalkyl group;

n is 0 or 1; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.In another more specific embodiment, said immunomodulatory compound is acompound having the structure

wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R is H or CH₂OCOR′;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹, R²,R³, or R⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbons

R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R′ is R⁷—CHR¹⁰—N(R⁸R⁹);

R⁷ is m-phenylene or p-phenylene or —(C_(n)H_(2n))— in which n has avalue of 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂X₁CH₂CH₂— in which X₁ is —O—,—S—, or —NH—;

R₁₀ is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

In another specific embodiment of the method, said solution additionallycomprises a vasodilator. In a more specific embodiment, said vasodilatoris an antihypertensive drug. In another more specific embodiment, saidvasodilator activates guanylyl cyclase, ADP-ribosyl transferase orcyclooxygenase, or inhibits lipoxygenase. In another more specificembodiment, said vasodilator is atrial natriuretic peptide (ANP),adrenocorticotropin, corticotropin-releasing hormone, sodiumnitroprusside, hydralazine, adenosine triphosphate, adenosine,indomethacin or magnesium sulfate. In a more specific embodiment, saidhydralazine is present in a concentration of from about 0.1 mM to about10 mM. In another more specific embodiment, said adenosine is present ata concentration of about 0.001 mM to about 10.0 mM. In another morespecific embodiment, said adenosine triphosphate is present at aconcentration of about 0.1 mM to about 1000 mM. In another more specificembodiment, said indomethacin is present at a concentration of about 1mg/kg to about 20 mg/kg, wherein “kg” is the weight of the placenta. Inanother more specific embodiment, said magnesium sulfate is present at aconcentration of about 0.1 mM to about 20 mM.

In another specific embodiment of the method, said stem cell is a CD34⁺stem cell. In a more specific embodiment, CD34⁺ stem cell is aCD34⁺CD38⁻ stem cell. In an more specific embodiment, said CD34⁺CD38⁻stem cell is part of a population of CD34⁺CD38-stem cells present inplacental perfusate as a higher percentage of total nucleated cells ascompared to the percentage of CD34⁺CD38⁻ cells in cord blood. In anotherspecific embodiment of the invention, said stem cell is a CD34⁻ stemcell. In a more specific embodiment, said CD34⁻ stem cell isadditionally CD31⁺, CD33⁺, CD44⁻, CD117⁺, KDR⁺, HLA-ABC^(weak), orHLA-DR^(weak). In another more specific embodiment, said CD34⁻ stem cellis additionally ABC-p⁺, SSEA3⁺, and SSEA4⁺.

The invention also provides compositions, e.g., solutions, thatfacilitate stem cell collection, for example, collection by perfusion,and/or by tissue disruption, e.g., enzymatic digestion. In oneembodiment, the invention provides a composition comprising, in aphysiologically-acceptable solution, an inhibitor of apoptosis and aprotease, wherein said composition, when contacted with population ofstem cells, reduces or prevents apoptosis in said population of stemcells as compared to a population of stem cells not contacted with thecomposition. In a specific embodiment, said composition is not anaturally-occurring composition. In another specific embodiment, saidinhibitor of apoptosis is a caspase inhibitor. In another specificembodiment, said inhibitor of apoptosis is a JNK inhibitor. In a morespecific embodiment, said JNK inhibitor does not modulatedifferentiation or proliferation of said stem cells. In another specificembodiment, said protease is present in an amount sufficient todetectably dissociate the cells of a tissue from which said stem cellsmay be derived. In another embodiment, the composition additionallycomprises an inhibitor of necrosis. In a more specific embodiment, saidinhibitor of necrosis is 2-(1H-Indol-3-yl)-3-pentylamino-maleimide. Inanother specific embodiment, the composition additionally comprises anoxygen-carrying perfluorocarbon. In another specific embodiment, saidphysiologically-acceptable solution is a saline solution or culturemedium. In a more specific embodiment, said saline solution is 0.9% NaClsolution or phosphate buffered saline. In another more specificembodiment, said protease is a matrix metalloprotease or a neutralprotease. In a more specific embodiment, said matrix metalloprotease iscollagenase. In another specific embodiment, said neutral protease isthermolysin or dispase. In another specific embodiment, the compositionadditionally comprises a mucolytic enzyme. In a more specificembodiment, said mucolytic enzyme is hyaluronidase.

In another specific embodiment, the composition additionally compriseshydroxyethyl starch, lactobionic acid and raffinose. In another specificembodiment, the composition additionally comprises UW solution.

In a specific embodiment of the composition, said JNK inhibitor is anindazole. In another specific embodiment, said JNK inhibitor has thestructure

wherein:

A is a direct bond, —(CH₂)_(a)—, —(CH₂)_(b)CH═CH(CH₂)_(c)—, or—(CH₂)_(b)C≡C(CH₂)_(c)—;

R₁ is aryl, heteroaryl or heterocycle fused to phenyl, each beingoptionally substituted with one to four substituents independentlyselected from R₃;

R₂ is —R₃, —R₄, —(CH₂)_(b)C(═O)R₅, —(CH₂)_(b)C(═O)OR₅,—(CH₂)_(b)C(═O)NR₅R₆, —(CH₂)_(b)C(═O)NR₅(CH₂)_(c)C(═O)R₆,—(CH₂)_(b)NR₅C(═O)R₆, —(CH₂)_(b)NR₅C(═O)NR₆R₇, —(CH₂)_(b)NR₅R₆,—(CH₂)_(b)OR₅, —(CH₂)_(b)SO_(d)R₅ or —(CH₂)_(b)SO₂NR₅R₆;

a is 1, 2, 3, 4, 5 or 6;

b and c are the same or different and at each occurrence independentlyselected from 0, 1, 2, 3 or 4;

d is at each occurrence 0, 1 or 2;

R₃ is at each occurrence independently halogen, hydroxy, carboxy, alkyl,alkoxy, haloalkyl, acyloxy, thioalkyl, sulfinylalkyl, sulfonylalkyl,hydroxyalkyl, aryl, arylalkyl, heterocycle, heterocycloalkyl, —C(═O)OR₈,—OC(═O)R₈, —C(═O)NR₉R₉, —C(═O)NR₉OR₉, —SO₂NR₈R₉, —NR₈SO₂R₉, —CN, —NO₂,—NR₈R₉, —NR₈C(═O)R₉, —NR₈C(═O)(CH₂)_(b)OR₉, —NR₈C(═O)(CH₂)_(b)R₉,—O(CH₂)_(b)NR₈R₉, or heterocycle fused to phenyl;

R₄ is alkyl, aryl, arylalkyl, heterocycle or heterocycloalkyl, eachbeing optionally substituted with one to four substituents independentlyselected from R₃, or R₄ is halogen or hydroxy;

R₅, R₆ and R₇ are the same or different and at each occurrenceindependently hydrogen, alkyl, aryl, arylalkyl, heterocycle orheterocycloalkyl, wherein each of R₅, R₆ and R₇ are optionallysubstituted with one to four substituents independently selected fromR₃; and

R₈ and R₉ are the same or different and at each occurrence independentlyhydrogen, alkyl, aryl, arylalkyl, heterocycle, or heterocycloalkyl, orR₈ and R₉ taken together with the atom or atoms to which they are bondedform a heterocycle, wherein each of R₈, R₉, and R₉ and R₉ taken togetherto form a heterocycle are optionally substituted with one to foursubstituents independently selected from R₃;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

In another specific embodiment of the composition, said JNK inhibitorhas the structure

wherein:

R₁ is aryl or heteroaryl optionally substituted with one to foursubstituents independently selected from R₇;

R₂ is hydrogen;

R₃ is hydrogen or lower alkyl;

R₄ represents one to four optional substituents, wherein eachsubstituent is the same or different and independently selected fromhalogen, hydroxy, lower alkyl and lower alkoxy;

R₅ and R₆ are the same or different and independently —R₈,—(CH₂)_(a)C(═O)R₉, —(CH₂)_(a)C(═O)OR₉, —(CH₂)_(a)C(═O)NR₉R₁₀,—(CH₂)_(a)C(═O)NR₉(CH₂)_(b)C(═O)R₁₀, —(CH₂)_(a)NR₉C(═O)R₁₀,(CH₂)_(a)NR₁₁C(═O)NR₉R₁₀, —(CH₂)_(a)NR₉R₁₀, —(CH₂)_(a)OR₉,—(CH₂)_(a)SO_(c)R₉ or —(CH₂)_(a)SO₂NR₉R₁₀; or R₅ and R₆ taken togetherwith the nitrogen atom to which they are attached to form a heterocycleor substituted heterocycle;

R₇ is at each occurrence independently halogen, hydroxy, cyano, nitro,carboxy, alkyl, alkoxy, haloalkyl, acyloxy, thioalkyl, sulfinylalkyl,sulfonylalkyl, hydroxyalkyl, aryl, arylalkyl, heterocycle, substitutedheterocycle, heterocycloalkyl, —C(═O)OR₈, —OC(═O)R₈, —C(═O)NR₈R₉,—C(═O)NR₈OR₉, —SO_(c)R₈, —SO_(c)NR₈R₉, —NR₈SO_(c)R₉, —NR₈R₉,—NR₈C(═O)R₉, —NR₈C(═O)(CH₂)_(b)OR₉, —NR₈C(═O)(CH₂)_(b)R₉,—O(CH₂)_(b)NR₈R₉, or heterocycle fused to phenyl;

R₈, R₉, R₁₀ and R₁₁ are the same or different and at each occurrenceindependently hydrogen, alkyl, aryl, arylalkyl, heterocycle,heterocycloalkyl; or R₈ and R₉ taken together with the atom or atoms towhich they are attached to form a heterocycle;

a and b are the same or different and at each occurrence independentlyselected from 0, 1, 2, 3 or 4; and

c is at each occurrence 0, 1 or 2;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

In another specific embodiment of the composition, said JNK inhibitorhas the structure

wherein R₀ is —O—, —S—, —S(O)—, —S(O)₂—, NH or —CH₂—;

the compound of structure (III) being: (i) unsubstituted, (ii)monosubstituted and having a first substituent, or (iii) disubstitutedand having a first substituent and a second substituent;

the first or second substituent, when present, is at the 3, 4, 5, 7, 8,9, or 10 position, wherein the first and second substituent, whenpresent, are independently alkyl, hydroxy, halogen, nitro,trifluoromethyl, sulfonyl, carboxyl, alkoxycarbonyl, alkoxy, aryl,aryloxy, arylalkyloxy, arylalkyl, cycloalkylalkyloxy, cycloalkyloxy,alkoxyalkyl, alkoxyalkoxy, aminoalkoxy, mono-alkylaminoalkoxy,di-alkylaminoalkoxy, or a group represented by structure (a), (b), (c),(d), (e), or (f):

wherein R₃ and R₄ are taken together and represent alkylidene or aheteroatom-containing cyclic alkylidene or R₃ and R₄ are independentlyhydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,aryloxyalkyl, alkoxyalkyl, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonylalkyl, amino, mono-alkylamino,di-alkylamino, arylamino, arylalkylamino, cycloalkylamino,cycloalkylalkylamino, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

In a more specific embodiment, the composition additionally comprises animmunomodulatory compound. In another more specific embodiment, saidimmunomodulatory compound is3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione;3-(4′aminoisolindoline-1′-onw)-1-piperidine-2,6-dione;4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; orα-(3-aminophthalimido) glutarimide. In another more specific embodiment,said immunomodulatory compound is a compound having the structure

wherein one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl, or a pharmaceutically acceptable salt,hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, ormixture of stereoisomers thereof. In another more specific embodiment,said immunomodulatory compound is a compound having the structure

wherein one of X and Y is C═O and the other is CH₂ or C═O;

R¹ is H, (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-C₂-C₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴,(C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-R⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³,C(S)NHR³, C(O)NR³R^(3′), C(S)NR³R^(3′) or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl;

R³ and R^(3′) are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-C₂-C₅)heteroaryl,(C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵,benzyl, aryl, (C₀-C₄)alkyl-C₁-C₆)heterocycloalkyl, or(C₀-C₄)alkyl-C₂-C₅)heteroaryl;

R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, or(C₂-C₅)heteroaryl; each occurrence of R⁶ is independently H,(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₂-C₅)heteroaryl, or (C₀-C₈)alkyl-C(O)O—R⁵ or the R⁶ groups can join toform a heterocycloalkyl group;

n is 0 or 1; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.In another more specific embodiment, said immunomodulatory compound is acompound having the structure

wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R is H or CH₂OCOR′;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹, R²,R³, or R⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbons

R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R′ is R⁷—CHR¹⁰—N(R⁸R⁹);

R⁷ is m-phenylene or p-phenylene or —(C_(n)H_(2n))— in which n has avalue of 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂X₁CH₂CH₂— in which X₁ is —O—,—S—, or —NH—;

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

In another specific embodiment, the composition additionally comprises avasodilator. In a more specific embodiment, said vasodilator is anantihypertensive drug. In another more specific embodiment, saidvasodilator activates guanylyl cyclase, ADP-ribosyl transferase orcyclooxygenase, or inhibits lipoxygenase. In another more specificembodiment, said vasodilator is atrial natriuretic peptide (ANP),adrenocorticotropin, corticotropin-releasing hormone, sodiumnitroprusside, hydralazine, adenosine triphosphate, adenosine,indomethacin or magnesium sulfate. In an even more specific embodiment,said hydralazine is present in a concentration of from about 0.1 mM toabout 10 mM. In another more specific embodiment, said adenosine ispresent at a concentration of about 0.001 mM to about 10.0 mM. Inanother more specific embodiment, said adenosine triphosphate is presentat a concentration of about 0.1 mM to about 1000 mM. In another morespecific embodiment, said indomethacin is present at a concentration ofabout 1 mg/kg to about 20 mg/kg, wherein “kg” is the weight of theplacenta. In another more specific embodiment, said magnesium sulfate ispresent at a concentration of about 0.1 mM to about 20 mM.

In another aspect, the invention provides a method of preserving apopulation of stem cells, comprising contacting a population of stemcells with an inhibitor of apoptosis and an oxygen-carryingperfluorocarbon, wherein said inhibitor of apoptosis is present in anamount and for a time sufficient to reduce or prevent apoptosis in thepopulation of stem cells, as compared to a population of stem cells notcontacted with the inhibitor of apoptosis. In a specific embodiment,said inhibitor of apoptosis is a caspase inhibitor. In another specificembodiment, said inhibitor of apoptosis is a JNK inhibitor. In a morespecific embodiment, said JNK inhibitor does not modulatedifferentiation or proliferation of said stem cells. In another morespecific embodiment, said JNK inhibitor and said perfluorocarbon arecontained within a single solution prior to said contacting. In anothermore specific embodiment, said JNK inhibitor is contained within a firstsolution, and said perfluorocarbon is contained within a secondsolution, prior to said contacting. In another specific embodiment, saidsolution, first solution, or second solution additionally compriseshydroxyethyl starch, lactobionic anion and raffinose. The method ofclaim 95, wherein said JNK inhibitor and said perfluorocarbon arebetween about 0° C. and about 25° C. at the time of said contacting. Inanother more specific embodiment, said JNK inhibitor and saidperfluorocarbon are between about 2° C. and 10° C., or between about 2°C. and about 5° C., at the time of said contacting. In another morespecific embodiment, said contacting is performed during transport ofsaid population of stem cells. In another more specific embodiment, saidcontacting is performed during freezing and thawing of said populationof stem cells.

In another specific embodiment, the method additionally comprisescontacting said population of stem cells with an inhibitor of necrosis.In a more specific embodiment, said inhibitor of necrosis is2-(1H-Indol-3-yl)-3-pentylamino-maleimide.

In another specific embodiment of the method, said population of stemcells is exposed to a hypoxic condition for less than 72 hours duringsaid preservation, wherein a hypoxic condition is a concentration ofoxygen that is less than normal blood oxygen concentration. In a morespecific embodiment, said population of stem cells is exposed to saidhypoxic condition for less than 48 hours during said preservation. Inanother more specific embodiment, said population of stem cells isexposed to said hypoxic condition for less than 24 hours, or less than 6hours, or is not exposed to a hypoxic condition, during saidpreservation. In another specific embodiment, said population of stemcells is not exposed to shear stress during said preservation.

In another specific embodiment, any of the foregoing solutions comprisesUW solution.

In another specific embodiment of the method, said population of stemcells is contained within, or isolated from, a placenta.

In a more specific embodiment of the method, said JNK inhibitor has thestructure

wherein:

A is a direct bond, —(CH₂)_(a)—, —(CH₂)_(b)CH═CH(CH₂)_(c)—, or—(CH₂)_(b)C≡C(CH₂)_(c)—;

R₁ is aryl, heteroaryl or heterocycle fused to phenyl, each beingoptionally substituted with one to four substituents independentlyselected from R₃;

R₂ is —R₃, —R₄, —(CH₂)_(b)C(═O)R₅, —(CH₂)_(b)C(═O)OR₅,—(CH₂)_(b)C(═O)NR₅R₆,

—(CH₂)_(b)C(═O)NR₅(CH₂)_(c)C(═O)R₆, —(CH₂)_(b)NR₅C(═O)R₆,—(CH₂)_(b)NR₅C(═O)NR₆R₇, —(CH₂)_(b)NR₅R₆, —(CH₂)_(b)OR₅,

—(CH₂)_(b)SO_(d)R₅ or —(CH₂)_(b)SO₂NR₅R₆;

a is 1, 2, 3, 4, 5 or 6;

b and c are the same or different and at each occurrence independentlyselected from 0, 1, 2, 3 or 4;

d is at each occurrence 0, 1 or 2;

R₃ is at each occurrence independently halogen, hydroxy, carboxy, alkyl,alkoxy, haloalkyl, acyloxy, thioalkyl, sulfinylalkyl, sulfonylalkyl,hydroxyalkyl, aryl, arylalkyl, heterocycle, heterocycloalkyl, —C(═O)OR₈,—OC(═O)R₈, —C(═O)NR₈R₉, —C(═O)NR₈OR₉, —SO₂NR₈R₉, —NR₈SO₂R₉, —CN, —NO₂,—NR₈R₉, —NR₈C(═O)R₉, —NR₈C(═O)(CH₂)_(b)OR₉, —NR₉C(═O)(CH₂)_(b)R₉,—O(CH₂)_(b)NR₉R₉, or heterocycle fused to phenyl;

R₄ is alkyl, aryl, arylalkyl, heterocycle or heterocycloalkyl, eachbeing optionally substituted with one to four substituents independentlyselected from R₃, or R₄ is halogen or hydroxy;

R₅, R₆ and R₇ are the same or different and at each occurrenceindependently hydrogen, alkyl, aryl, arylalkyl, heterocycle orheterocycloalkyl, wherein each of R₅, R₆ and R₇ are optionallysubstituted with one to four substituents independently selected fromR₃; and

R₈ and R₉ are the same or different and at each occurrence independentlyhydrogen, alkyl, aryl, arylalkyl, heterocycle, or heterocycloalkyl, orR₈ and R₉ taken together with the atom or atoms to which they are bondedform a heterocycle, wherein each of R₈, R₉, and R₈ and R₉ taken togetherto form a heterocycle are optionally substituted with one to foursubstituents independently selected from R₃.

In another more specific embodiment of the method, said JNK inhibitorhas the structure

wherein:

R₁ is aryl or heteroaryl optionally substituted with one to foursubstituents independently selected from R₇;

R₂ is hydrogen;

R₃ is hydrogen or lower alkyl;

R₄ represents one to four optional substituents, wherein eachsubstituent is the same or different and independently selected fromhalogen, hydroxy, lower alkyl and lower alkoxy;

R₅ and R₆ are the same or different and independently —R₈,—(CH₂)_(a)C(═O)R₉, —(CH₂)_(a)C(═O)OR₉, —(CH₂)_(a)C(═O)NR₉R₁₀,—(CH₂)_(a)C(═O)NR₉(CH₂)_(b)C(═O)R₁₀, —(CH₂)_(a)NR₉C(═O)R₁₀,(CH₂)_(a)NR₁₁C(═O)NR₉R₁₀, —(CH₂)_(a)NR₉R₁₀, —(CH₂)_(a)OR₉,—(CH₂)_(a)SO_(c)R₉ or —(CH₂)_(a)SO₂NR₉R₁₀;

or R₅ and R₆ taken together with the nitrogen atom to which they areattached to form a heterocycle or substituted heterocycle;

R₇ is at each occurrence independently halogen, hydroxy, cyano, nitro,carboxy, alkyl, alkoxy, haloalkyl, acyloxy, thioalkyl, sulfinylalkyl,sulfonylalkyl, hydroxyalkyl, aryl, arylalkyl, heterocycle, substitutedheterocycle, heterocycloalkyl, —C(═O)OR₈, —OC(═O)R₈, —C(═O)NR₈R₉,—C(═O)NR₈OR₉, —SO_(c)R₈, —SO_(c)NR₈R₉, —NR₈SO_(c)R₉, —NR₈R₉,—NR₈C(═O)R₉, —NR₈C(═O)(CH₂)_(b)OR₉, —NR₉C(═O)(CH₂)_(b)R₉,—O(CH₂)_(b)NR₈R₉, or heterocycle fused to phenyl;

R₈, R₉, R₁₀ and R₁₁ are the same or different and at each occurrenceindependently hydrogen, alkyl, aryl, arylalkyl, heterocycle,heterocycloalkyl;

or R₈ and R₉ taken together with the atom or atoms to which they areattached to form a heterocycle;

a and b are the same or different and at each occurrence independentlyselected from 0, 1, 2, 3 or 4; and

c is at each occurrence 0, 1 or 2.

In another more specific embodiment of the method, said JNK inhibitorhas the structure

wherein R₀ is —O—, —S—, —S(O)—, —S(O)₂—, NH or —CH₂—;

the compound of structure (III) being: (i) unsubstituted, (ii)monosubstituted and having a first substituent, or (iii) disubstitutedand having a first substituent and a second substituent;

the first or second substituent, when present, is at the 3, 4, 5, 7, 8,9, or 10 position, wherein the first and second substituent, whenpresent, are independently alkyl, hydroxy, halogen, nitro,trifluoromethyl, sulfonyl, carboxyl, alkoxycarbonyl, alkoxy, aryl,aryloxy, arylalkyloxy, arylalkyl, cycloalkylalkyloxy, cycloalkyloxy,alkoxyalkyl, alkoxyalkoxy, aminoalkoxy, mono-alkylaminoalkoxy,di-alkylaminoalkoxy, or a group represented by structure (a), (b), (c),(d), (e), or (f):

wherein R₃ and R₄ are taken together and represent alkylidene or aheteroatom-containing cyclic alkylidene or R₃ and R₄ are independentlyhydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,aryloxyalkyl, alkoxyalkyl, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonylalkyl, amino, mono-alkylamino,di-alkylamino, arylamino, arylalkylamino, cycloalkylamino,cycloalkylalkylamino, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl.

In an even more specific embodiment, the method additionally comprisescontacting the stem cells with an immunomodulatory compound. In a morespecific embodiment, said immunomodulatory compound is3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione;3-(4′aminoisolindoline-1′-onw)-1-piperidine-2,6-dione;4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; orα-(3-aminophthalimido) glutarimide. In another more specific embodiment,said immunomodulatory compound is a compound having the structure

wherein one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl, or a pharmaceutically acceptable salt,hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, ormixture of stereoisomers thereof. In another more specific embodiment,said immunomodulatory compound is a compound having the structure

wherein one of X and Y is C═O and the other is CH₂ or C═O;

R¹ is H, (C—C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-C₂-C₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴,(C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³,C(S)NHR³, C(O)NR³R^(3′), C(S)NR³R^(3′) or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl;

R³ and R^(3′) are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-C₂-C₅)heteroaryl,(C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵,benzyl, aryl, (C₀-C₄)alkyl-C₁-C₆)heterocycloalkyl, or(C₀-C₄)alkyl-C₂-C₅)heteroaryl;

R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, or(C₂-C₅)heteroaryl; each occurrence of R⁶ is independently H,(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₂-C₅)heteroaryl, or (C₀-C₈)alkyl-C(O)O—R⁵ or the R⁶ groups can join toform a heterocycloalkyl group;

n is 0 or 1; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.In another more specific embodiment, said immunomodulatory compound is acompound having the structure

wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R is H or CH₂OCOR′;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹, R²,R³, or R⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbons

R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R′ is R⁷—CHR¹⁰—N(R⁸R⁹);

R⁷ is m-phenylene or p-phenyle2ne or —(C_(n)H_(2n))— in which n has avalue of 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂X₁CH₂CH₂— in which X₁ is —O—,—S—, or —NH—;

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

In another specific embodiment of any of the above methods orcompositions, said JNK inhibitor is present at a concentration of fromabout 0.5 μM to about 10 μM. In another specific embodiment of the abovemethods or compositions, said protease is present at a concentration ofabout 0.1 mg/mL to about 10 mg/mL. In another specific embodiment of theabove methods or compositions, said mucolytic enzyme is present at aconcentration of from about 0.1 mg/mL to about 10 mg/mL. In anotherspecific embodiment of the above methods or compositions, saidimmunomodulatory compound is present at a concentration of from about0.5 μM to about 10 μM. In another specific embodiment of the abovemethods or compositions, said vasodilator is atrial natriuretic peptide(ANP), adrenocorticotropin, corticotropin-releasing hormone, sodiumnitroprusside, hydralazine, adenosine triphosphate, adenosine,indomethacin or magnesium sulfate. In an even more specific embodiment,said hydralazine is present in a concentration of from about 0.1 mM toabout 10 mM. In another more specific embodiment, said adenosine ispresent at a concentration of about 0.001 mM to about 10.0 mM. Inanother more specific embodiment, said adenosine triphosphate is presentat a concentration of about 0.1 mM to about 1000 mM.

In another specific embodiment, any of the compositions described hereincan comprise a physiologically-acceptable solution comprising sodiumchloride, potassium chloride, magnesium sulfate, calcium chloride,sodium sulfate, potassium sulfate, sodium carbonate, and glucose. Any ofthe composition can comprise one or more essential or non-essentialamino acids, alone or in combination.

3.1 Definitions

As used herein, the term “embryonic stem cell” refers to a cell that isderived from the inner cell mass of a blastocyst (e.g., a 4- to5-day-old human embryo) and that is pluripotent.

As used herein, the term “placental stem cell” refers to a stem cell orprogenitor cell that is derived from a mammalian placenta, regardless ofcellular morphology, cell surface markers, or the number of passagesafter a primary culture. The term does not, however, encompass stemcells derived solely from another tissue, e.g., placental blood orumbilical cord blood.

As used herein, the term “exsanguinated” or “exsanguination,” when usedwith respect to the placenta, refers to the removal and/or draining ofsubstantially all cord blood from the placenta.

As used herein, the term “perfusate” refers to the fluid collectedfollowing its passage through an organ or tissue. In a preferredembodiment, the perfusate contains one or more anticoagulants.

As used herein, the term “stem cell” encompasses pluripotent andmultipotent cells, and progenitor cells, including committed progenitorcells. Multipotent and pluripotent stem cells retain the ability toproliferate and expand in culture.

“Alkyl” means a saturated straight chain or branched non-cyclichydrocarbon having from 1 to 10 carbon atoms. “Lower alkyl” means alkyl,as defined above, having from 1 to 4 carbon atoms. Representativesaturated straight chain alkyls include -methyl, -ethyl, -n-propyl,-n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl and-n-decyl; while saturated branched alkyls include -isopropyl,-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl,3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl,2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl,2,3-dimethylbutyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl,2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl,2,2-dimethylpentyl, 2,2-dimethylhexyl, 3,3-dimtheylpentyl,3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl,2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl,2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl,2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl,3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl and the like.

An “alkenyl group” or “alkylidene” mean a straight chain or branchednon-cyclic hydrocarbon having from 2 to 10 carbon atoms and including atleast one carbon-carbon double bond. Representative straight chain andbranched (C₂-C₁₀)alkenyls include -vinyl, -allyl, -1-butenyl,-2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl,-3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl,-1-hexenyl, -2-hexenyl, -3-hexenyl, -1-heptenyl, -2-heptenyl,-3-heptenyl, -1-octenyl, -2-octenyl, -3-octenyl, -1-nonenyl, -2-nonenyl,-3-nonenyl, -1-decenyl, -2-decenyl, -3-decenyl and the like. An alkenylgroup can be unsubstituted or substituted. A “cyclic alkylidene” is aring having from 3 to 8 carbon atoms and including at least onecarbon-carbon double bond, wherein the ring can have from 1 to 3heteroatoms.

An “alkynyl group” means a straight chain or branched non-cyclichydrocarbon having from 2 to 10 carbon atoms and including at lease onecarbon-carbon triple bond. Representative straight chain and branched—(C₂-C₁₀)alkynyls include -acetylenyl, -propynyl, -1-butynyl,-2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1-butynyl, -4-pentynyl,-1-hexynyl, -2-hexynyl, -5-hexynyl, -1-heptynyl, -2-heptynyl,-6-heptynyl, -1-octynyl, -2-octynyl, -7-octynyl, -1-nonynyl, -2-nonynyl,-8-nonynyl, -1-decynyl, -2-decynyl, -9-decynyl, and the like. An alkynylgroup can be unsubstituted or substituted.

The terms “Halogen” and “Halo” mean fluorine, chlorine, bromine oriodine.

“Haloalkyl” means an alkyl group, wherein alkyl is defined above,substituted with one or more halogen atoms.

“Keto” means a carbonyl group (i.e., C═O).

“Acyl” means an —C(O)alkyl group, wherein alkyl is defined above,including —C(O)CH₃, —C(O)CH₂CH₃, —C(O)(CH₂)₂CH₃,—C(O)(CH₂)₃CH₃—C(O)(CH₂)₄CH₃, —C(O)(CH₂)₅CH₃, and the like.

“Acyloxy” means an —OC(O)alkyl group, wherein alkyl is defined above,including —OC(O)CH₃, —OC(O)CH₂CH₃, —OC(O)(CH₂)₂CH₃, —OC(O)(CH₂)₃CH₃,—OC(O)(CH₂)₄CH₃, —OC(O)(CH₂)₅CH₃, and the like.

“Ester” means and —C(O)Oalkyl group, wherein alkyl is defined above,including —C(O)OCH₃, —C(O)OCH₂CH₃, —C(O)O(CH₂)₂CH₃, —C(O)O(CH₂)₃CH₃,—C(O)O(CH₂)₄CH₃, —C(O)O(CH₂)₅CH₃, and the like.

“Alkoxy” means —O-(alkyl), wherein alkyl is defined above, including—OCH₃, —OCH₂CH₃, —O(CH₂)₂CH₃, —O(CH₂)₃CH₃, —O(CH₂)₄CH₃, —O(CH₂)₅CH₃, andthe like. “Lower alkoxy” means —O-(lower alkyl), wherein lower alkyl isas described above.

“Alkoxyalkoxy” means —O-(alkyl)-O-(alkyl), wherein each alkyl isindependently an alkyl group defined above, including —OCH₂OCH₃,—OCH₂CH₂OCH₃, —OCH₂CH₂OCH₂CH₃, and the like.

“Alkoxycarbonyl” means —C(═O)O-(alkyl), wherein alkyl is defined above,including —C(═O)O—CH₃, —C(═O)O—CH₂CH₃, —C(═O)O—(CH₂)₂CH₃,—C(═O)O—(CH₂)₃CH₃, —C(═O)O—(CH₂)₄CH₃, —C(═O)O—(CH₂)₅CH₃, and the like.

“Alkoxycarbonylalkyl” means -(alkyl)-C(═O)O-(alkyl), wherein each alkylis independently defined above, including —CH₂—C(═O)O—CH₃,—CH₂—C(═O)O—CH₂CH₃, —CH₂—C(═O)O—(CH₂)₂CH₃, —CH₂—C(═O)O—(CH₂)₃CH₃,—CH₂—C(═O)O—(CH₂)₄CH₃, —CH₂—C(═O)O—(CH₂)SCH₃, and the like.

“Alkoxyalkyl” means -(alkyl)-O-(alkyl), wherein each alkyl isindependently an alkyl group defined above, including —CH₂OCH₃,—CH₂OCH₂CH₃, —(CH₂)₂OCH₂CH₃, —(CH₂)₂O(CH₂)₂CH₃, and the like.

“Aryl” means a carbocyclic aromatic group containing from 5 to 10 ringatoms. Representative examples include, but are not limited to, phenyl,tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, pyridinyl andnaphthyl, as well as benzo-fused carbocyclic moieties including5,6,7,8-tetrahydronaphthyl. A carbocyclic aromatic group can beunsubstituted or substituted. In one embodiment, the carbocyclicaromatic group is a phenyl group.

“Aryloxy” means —O-aryl group, wherein aryl is as defined above. Anaryloxy group can be unsubstituted or substituted. In one embodiment,the aryl ring of an aryloxy group is a phenyl group.

“Arylalkyl” means -(alkyl)-(aryl), wherein alkyl and aryl are as definedabove, including —(CH₂)phenyl, —(CH₂)₂phenyl, —(CH₂)₃phenyl,—CH(phenyl)₂, —CH(phenyl)₃, —(CH₂)tolyl, —(CH₂)anthracenyl,—(CH₂)fluorenyl, —(CH₂)indenyl, —(CH₂)azulenyl, —(CH₂)pyridinyl,—(CH₂)naphthyl, and the like.

“Arylalkyloxy” means —O-(alkyl)-(aryl), wherein alkyl and aryl aredefined above, including —O—(CH₂)₂phenyl, —O—(CH₂)₃phenyl,—O—CH(phenyl)₂, —O—CH(phenyl)₃, —O—(CH₂)tolyl, —O—(CH₂)anthracenyl,—O—(CH₂)fluorenyl, —O—(CH₂)indenyl, —O—(CH₂)azulenyl, —O—(CH₂)pyridinyl,—O—(CH₂)naphthyl, and the like.

“Aryloxyalkyl” means -(alkyl)-O-(aryl), wherein alkyl and aryl aredefined above, including —CH₂—O-(phenyl), —(CH₂)₂—O-phenyl,—(CH₂)₃—O-phenyl, —(CH₂)—O-tolyl, —(CH₂)—O-anthracenyl,—(CH₂)—O-fluorenyl, —(CH₂)—O-indenyl, —(CH₂)—O-azulenyl,—(CH₂)—O-pyridinyl, —(CH₂)—O-naphthyl, and the like.

“Cycloalkyl” means a monocyclic or polycyclic saturated ring havingcarbon and hydrogen atoms and having no carbon-carbon multiple bonds.Examples of cycloalkyl groups include, but are not limited to,(C₃-C₇)cycloalkyl groups, including cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and cycloheptyl, and saturated cyclic andbicyclic terpenes. A cycloalkyl group can be unsubstituted orsubstituted. In one embodiment, the cycloalkyl group is a monocyclicring or bicyclic ring.

“Cycloalkyloxy” means —O-(cycloalkyl), wherein cycloalkyl is definedabove, including —O-cyclopropyl, —O-cyclobutyl, —O-cyclopentyl,—O-cyclohexyl, —O-cycloheptyl and the like.

“Cycloalkylalkyloxy” means —O-(alkyl)-(cycloalkyl), wherein cycloalkyland alkyl are defined above, including —O—CH₂-cyclopropyl,—O—(CH₂)₂-cyclopropyl, —O—(CH₂)₃-cyclopropyl, —O—(CH₂)₄-cyclopropyl,O—CH₂-cyclobutyl, O—CH₂-cyclopentyl, O—CH₂-cyclohexyl,O—CH₂-cycloheptyl, and the like.

“Aminoalkoxy” means —O-(alkyl)-NH₂, wherein alkyl is defined above, suchas —O—CH₂—NH₂, —O—(CH₂)₂—NH₂, —O—(CH₂)₃—NH₂, —O—(CH₂)₄—NH₂,—O—(CH₂)₅—NH₂, and the like.

“Mono-alkylamino” means —NH(alkyl), wherein alkyl is defined above, suchas —NHCH₃, —NHCH₂CH₃, —NH(CH₂)₂CH₃, —NH(CH₂)₃CH₃, —NH(CH₂)₄CH₃,—NH(CH₂)₅CH₃, and the like.

“Di-alkylamino” means —N(alkyl)(alkyl), wherein each alkyl isindependently an alkyl group defined above, including —N(CH₃)₂,—N(CH₂CH₃)₂, —N((CH₂)₂CH₃)₂, —N(CH₃)(CH₂CH₃), and the like.

“Mono-alkylaminoalkoxy” means —O-(alkyl)-NH(alkyl), wherein each alkylis independently an alkyl group defined above, including —O—(CH₂)—NHCH₃,—O—(CH₂)—NHCH₂CH₃, —O—(CH₂)—NH(CH₂)₂CH₃, —O—(CH₂)—NH(CH₂)₃CH₃,—O—(CH₂)—NH(CH₂)₄CH₃, —O—(CH₂)—NH(CH₂)₅CH₃, —O—(CH₂)₂—NHCH₃, and thelike.

“Di-alkylaminoalkoxy” means —O-(alkyl)-N(alkyl)(alkyl), wherein eachalkyl is independently an alkyl group defined above, including—O—(CH₂)—N(CH₃)₂, —O—(CH₂)—N(CH₂CH₃)₂, —O—(CH₂)—N((CH₂)₂CH₃)₂,—O—(CH₂)—N(CH₃)(CH₂CH₃), and the like.

“Arylamino” means —NH(aryl), wherein aryl is defined above, including—NH(phenyl), —NH(tolyl), —NH(anthracenyl), —NH(fluorenyl), —NH(indenyl),—NH(azulenyl), —NH(pyridinyl), —NH(naphthyl), and the like.

“Arylalkylamino” means —NH-(alkyl)-(aryl), wherein alkyl and aryl aredefined above, including —NH—CH₂-(phenyl), —NH—CH₂-(tolyl),—NH—CH₂-(anthracenyl), —NH—CH₂-(fluorenyl), —NH—CH₂-(indenyl),—NH—CH₂-(azulenyl), —NH—CH₂-(pyridinyl), —NH—CH₂-(naphthyl),—NH—(CH₂)₂-(phenyl) and the like.

“Alkylamino” means mono-alkylamino or di-alkylamino as defined above,such as —N(alkyl)(alkyl), wherein each alkyl is independently an alkylgroup defined above, including —N(CH₃)₂, —N(CH₂CH₃)₂, —N((CH₂)₂CH₃)₂,—N(CH₃)(CH₂CH₃) and —N(alkyl)(alkyl), wherein each alkyl isindependently an alkyl group defined above, including —N(CH₃)₂,—N(CH₂CH₃)₂, —N((CH₂)₂CH₃)₂, —N(CH₃)(CH₂CH₃) and the like.

“Cycloalkylamino” means —NH-(cycloalkyl), wherein cycloalkyl is asdefined above, including —NH-cyclopropyl, —NH-cyclobutyl,—NH-cyclopentyl, —NH-cyclohexyl, —NH-cycloheptyl, and the like.

“Carboxyl” and “carboxy” mean —COOH.

“Cycloalkylalkylamino” means —NH-(alkyl)-(cycloalkyl), wherein alkyl andcycloalkyl are defined above, including —NH—CH₂-cyclopropyl,—NH—CH₂-cyclobutyl, —NH—CH₂-cyclopentyl, —NH—CH₂-cyclohexyl,—NH—CH₂-cycloheptyl, —NH—(CH₂)₂-cyclopropyl and the like.

“Aminoalkyl” means -(alkyl)-NH₂, wherein alkyl is defined above,including CH₂—NH₂, —(CH₂)₂—NH₂, —(CH₂)₃—NH₂, —(CH₂)₄—NH₂, —(CH₂)₅—NH₂and the like.

“Mono-alkylaminoalkyl” means -(alkyl)-NH(alkyl), wherein each alkyl isindependently an alkyl group defined above, including —CH₂—NH—CH₃,—CH₂—NHCH₂CH₃, —CH₂—NH(CH₂)₂CH₃, —CH₂—NH(CH₂)₃CH₃, —CH₂—NH(CH₂)₄CH₃,—CH₂—NH(CH₂)₅CH₃, —(CH₂)₂—NH—CH₃, and the like.

“Di-alkylaminoalkyl” means -(alkyl)-N(alkyl)(alkyl), wherein each alkylis independently an alkyl group defined above, including —CH₂—N(CH₃)₂,—CH₂—N(CH₂CH₃)₂, —CH₂—N((CH₂)₂CH₃)₂, —CH₂—N(CH₃)(CH₂CH₃),—(CH₂)₂—N(CH₃)₂, and the like.

“Heteroaryl” means an aromatic heterocycle ring of 5- to 10 members andhaving at least one heteroatom selected from nitrogen, oxygen andsulfur, and containing at least 1 carbon atom, including both mono- andbicyclic ring systems. Representative heteroaryls are triazolyl,tetrazolyl, oxadiazolyl, pyridyl, furyl, benzofuranyl, thiophenyl,benzothiophenyl, quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl,imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl,pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,cinnolinyl, phthalazinyl, quinazolinyl, pyrimidyl, oxetanyl, azepinyl,piperazinyl, morpholinyl, dioxanyl, thietanyl and oxazolyl.

“Heteroarylalkyl” means -(alkyl)-(heteroaryl), wherein alkyl andheteroaryl are defined above, including —CH₂-triazolyl, —CH₂-tetrazolyl,—CH₂-oxadiazolyl, —CH₂-pyridyl, —CH₂-furyl, —CH₂-benzofuranyl,—CH₂-thiophenyl, —CH₂-benzothiophenyl, —CH₂-quinolinyl, —CH₂-pyrrolyl,—CH₂-indolyl, —CH₂-oxazolyl, —CH₂-benzoxazolyl, —CH₂-imidazolyl,—CH₂-benzimidazolyl, —CH₂-thiazolyl, —CH₂-benzothiazolyl,—CH₂-isoxazolyl, —CH₂-pyrazolyl, —CH₂-isothiazolyl, —CH₂-pyridazinyl,—CH₂-pyrimidinyl, —CH₂-pyrazinyl, —CH₂-triazinyl, —CH₂-cinnolinyl,—CH₂-phthalazinyl, —CH₂-quinazolinyl, —CH₂-pyrimidyl, —CH₂-oxetanyl,—CH₂-azepinyl, —CH₂-piperazinyl, —CH₂-morpholinyl, —CH₂-dioxanyl,—CH₂-thietanyl, —CH₂-oxazolyl, —(CH₂)₂-triazolyl, and the like.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, andwhich contains from 1 to 4 heteroatoms independently selected fromnitrogen, oxygen and sulfur, and wherein the nitrogen and sulfurheteroatoms can be optionally oxidized, and the nitrogen heteroatom canbe optionally quaternized, including bicyclic rings in which any of theabove heterocycles are fused to a benzene ring. The heterocycle can beattached via any heteroatom or carbon atom. Heterocycles includeheteroaryls as defined above. Representative heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl,valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

“Heterocycle fused to phenyl” means a heterocycle, wherein heterocycleis defined as above, that is attached to a phenyl ring at two adjacentcarbon atoms of the phenyl ring.

“Heterocycloalkyl” means -(alkyl)-(heterocycle), wherein alkyl andheterocycle are defined above, including —CH₂-morpholinyl,—CH₂-pyrrolidinonyl, —CH₂-pyrrolidinyl, —CH₂-piperidinyl,—CH₂-hydantoinyl, —CH₂-valerolactamyl, —CH₂-oxiranyl, —CH₂-oxetanyl,—CH₂-tetrahydrofuranyl, —CH₂-tetrahydropyranyl,—CH₂-tetrahydropyridinyl, —CH₂-tetrahydroprimidinyl,—CH₂-tetrahydrothiophenyl, —CH₂-tetrahydrothiopyranyl,—CH₂-tetrahydropyrimidinyl, —CH₂-tetrahydrothiophenyl,—CH₂-tetrahydrothiopyranyl, and the like.

The term “substituted” as used herein means any of the above groups(i.e., aryl, arylalkyl, heterocycle and heterocycloalkyl) wherein atleast one hydrogen atom of the moiety being substituted is replaced witha substituent. In one embodiment, each carbon atom of the group beingsubstituted is substituted with no more that two substituents. Inanother embodiment, each carbon atom of the group being substituted issubstituted with no more than one substituent. In the case of a ketosubstituent, two hydrogen atoms are replaced with an oxygen which isattached to the carbon via a double bond. Substituents include halogen,hydroxyl, alkyl, haloalkyl, mono- or di-substituted aminoalkyl,alkyloxyalkyl, aryl, arylalkyl, heterocycle, heterocycloalkyl,—NR_(a)R_(b), —NR_(a)C(═O)R_(b), —NR_(a)C(═O)NR_(a)R_(b),—NR_(a)C(═O)OR_(b), —NR_(a)SO₂R_(b), —OR_(a), —C(═O)R_(a), —C(═O)OR_(a),—C(═O)NR_(a)R_(b), —OC(═O)R_(a), —OC(═O)OR_(a), —OC(═O)NR_(a)R_(b),—NR_(a)SO₂R_(b), or a radical of the formula -Y-Z-R_(a) where Y isalkanediyl, or a direct bond, Z is —O—, —S—, —N(R_(b))—, —C(═O)—,—C(═O)O—, —OC(═O)—, —N(R_(b))C(═O)—, —C(═O)N(R_(b))— or a direct bond,wherein R_(a) and R_(b) are the same or different and independentlyhydrogen, amino, alkyl, haloalkyl, aryl, arylalkyl, heterocycle, orheterocylealkyl, or wherein R_(a) and R_(b) taken together with thenitrogen atom to which they are attached form a heterocycle.

“Haloalkyl” means alkyl, wherein alkyl is defined as above, having oneor more hydrogen atoms replaced with halogen, wherein halogen is asdefined above, including —CF₃, —CHF₂, —CH₂F, —CBr₃, —CHBr₂, —CH₂Br,—CCl₃, —CHCl₂, —CH₂Cl, —Cl₃, —CHI₂, —CH₂I, —CH₂—CF₃, —CH₂—CHF₂,—CH₂—CH₂F, —CH₂—CBr₃, —CH₂—CHBr₂, —CH₂—CH₂Br, —CH₂—CCl₃, —CH₂—CHCl₂,—CH₂—CH₂Cl, —CH₂—Cl₃, —CH₂—CHI₂, —CH₂—CH₂I, and the like.

“Hydroxyalkyl” means alkyl, wherein alkyl is as defined above, havingone or more hydrogen atoms replaced with hydroxy, including —CH₂OH,—CH₂CH₂OH, —(CH₂)₂CH₂OH, —(CH₂)₃CH₂OH, —(CH₂)₄CH₂OH, —(CH₂)₅CH₂OH,—CH(OH)—CH₃, —CH₂CH(OH)CH₃, and the like.

“Hydroxy” means —OH.

“Sulfonyl” means —SO₃H.

“Sulfonylalkyl” means —SO₂-(alkyl), wherein alkyl is defined above,including —SO₂—CH₃, —SO₂—CH₂CH₃, —SO₂—(CH₂)₂CH₃, —SO₂—(CH₂)₃CH₃,—SO₂—(CH₂)₄CH₃, —SO₂—(CH₂)₅CH₃, and the like.

“Sulfinylalkyl” means —SO-(alkyl), wherein alkyl is defined above,including —SO—CH₃, —SO—CH₂CH₃, —SO—(CH₂)₂CH₃, —SO—(CH₂)₃CH₃,—SO—(CH₂)₄CH₃, —SO—(CH₂)₅CH₃, and the like.

“Sulfonamidoalkyl” means —NHSO₂-(alkyl), wherein alkyl is defined above,including —NHSO₂—CH₃, —NHSO₂—CH₂CH₃, —NHSO₂—(CH₂)₂CH₃, —NHSO₂—(CH₂)₃CH₃,—NHSO₂—(CH₂)₄CH₃, —NHSO₂—(CH₂)₅CH₃, and the like.

“Thioalkyl” means —S-(alkyl), wherein alkyl is defined above, including—S—CH₃, —S—CH₂CH₃, —S—(CH₂)₂CH₃, —S—(CH₂)₃CH₃, —S—(CH₂)₄CH₃,—S—(CH₂)₅CH₃, and the like.

As used herein, the term “JNK inhibitor(s)” encompasses, but is notlimited to, compounds disclosed herein. A JNK inhibitor can be in theform of a pharmaceutically acceptable salt, free base, solvate, hydrate,stereoisomer, clathrate or prodrug thereof. Such inhibitory activity canbe determined by an assay or animal model well-known in the artincluding those set forth in Section 5. In one embodiment, the JNKinhibitor is a compound of structure (I)-(III).

As used herein and unless otherwise indicated, the term“pharmaceutically acceptable salt” encompasses non-toxic acid and baseaddition salts of the compound to which the term refers. Acceptablenon-toxic acid addition salts include those derived from organic andinorganic acids or bases known in the art, which include, for example,hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid,methanesulphonic acid, acetic acid, tartaric acid, lactic acid, succinicacid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid,salicylic acid, phthalic acid, embolic acid, enanthic acid, and thelike.

Compounds that are acidic in nature are capable of forming salts withvarious pharmaceutically acceptable bases. The bases that can be used toprepare pharmaceutically acceptable base addition salts of such acidiccompounds are those that form non-toxic base addition salts, i.e., saltscontaining pharmacologically acceptable cations such as, but not limitedto, alkali metal or alkaline earth metal salts and the calcium,magnesium, sodium or potassium salts in particular. Suitable organicbases include, but are not limited to, N,N-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumaine(N-methylglucamine), lysine, and procaine.

As used herein and unless otherwise indicated, the term “prodrug” meansa derivative of a compound that can hydrolyze, oxidize, or otherwisereact under biological conditions (in vitro or in vivo) to provide thecompound. Examples of prodrugs include, but are not limited to,derivatives of JNK inhibitors that comprise biohydrolyzable moietiessuch as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzablecarbamates, biohydrolyzable carbonates, biohydrolyzable ureides, andbiohydrolyzable phosphate analogues. Other examples of prodrugs includederivatives of a JNK inhibitor that comprise —NO, —NO₂, —ONO, or —ONO₂moieties. Prodrugs can typically be prepared using well-known methods,such as those described in 1 Burger's Medicinal Chemistry and DrugDiscovery, 172-178, 949-982 (Manfred E. Wolff ed., 5th ed. 1995), andDesign of Prodrugs (H. Bundgaard ed., Elselvier, N.Y. 1985).

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide,” “biohydrolyzable ester,” “biohydrolyzablecarbamate,” “biohydrolyzable carbonate,” “biohydrolyzable ureide,” and“biohydrolyzable phosphate” mean an amide, ester, carbamate, carbonate,ureide, or phosphate, respectively, of a compound that either: 1) doesnot interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, lower acyloxyalkyl esters (such as acetoxylmethyl,acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyl, andpivaloyloxyethyl esters), lactonyl esters (such as phthalidyl andthiophthalidyl esters), lower alkoxyacyloxyalkyl esters (such asmethoxycarbonyloxymethyl, ethoxycarbonyloxyethyl andisopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters,and acylamino alkyl esters (such as acetamidomethyl esters). Examples ofbiohydrolyzable amides include, but are not limited to, lower alkylamides, a-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides. Examples of biohydrolyzable carbamatesinclude, but are not limited to, lower alkylamines, substitutedethylenediamines, aminoacids, hydroxyalkylamines, heterocyclic andheteroaromatic amines, and polyether amines.

Various JNK inhibitors and immunomodulatory compounds contain one ormore chiral centers, and can exist as racemic mixtures of enantiomers ormixtures of diastereomers. This invention encompasses the use ofstereomerically pure forms of such compounds, as well as the use ofmixtures of those forms. For example, mixtures comprising equal orunequal amounts of the enantiomers of JNK inhibitors or immunomodulatorycompounds may be used in methods and compositions of the invention. Thepurified (R) or (S) enantiomers of the specific compounds disclosedherein may be used substantially free of its other enantiomer.

As used herein and unless otherwise indicated, the term “stereomericallypure” means a composition that comprises one stereoisomer of a compoundand is substantially free of other stereoisomers of that compound. Forexample, a stereomerically pure composition of a compound having onechiral center will be substantially free of the opposite enantiomer ofthe compound. A stereomerically pure composition of a compound havingtwo chiral centers will be substantially free of other diastereomers ofthe compound. A typical stereomerically pure compound comprises greaterthan about 80% by weight of one stereoisomer of the compound and lessthan about 20% by weight of other stereoisomers of the compound, morepreferably greater than about 90% by weight of one stereoisomer of thecompound and less than about 10% by weight of the other stereoisomers ofthe compound, even more preferably greater than about 95% by weight ofone stereoisomer of the compound and less than about 5% by weight of theother stereoisomers of the compound, and most preferably greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound.

As used herein and unless otherwise indicated, the term “stereomericallyenriched” means a composition that comprises greater than about 60% byweight of one stereoisomer of a compound, preferably greater than about70% by weight, more preferably greater than about 80% by weight of onestereoisomer of a compound.

As used herein and unless otherwise indicated, the term“enantiomerically pure” means a stereomerically pure composition of acompound having one chiral center. Similarly, the term “enantiomericallyenriched” means a stereomerically enriched composition of a compoundhaving one chiral center.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a cross-sectional view of the cannulation of the vein andartery of a placenta to perfuse the placenta and then collect theperfusate.

FIGS. 2 a-e are schematics showing the collection, clamping, perfusion,collection and storage of an exsanguinated and perfused placenta.

FIG. 3 is a cross-sectional schematic of a perfused placenta in a devicefor use as a bioreactor.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 Stem Cell CollectionCompositions

In one aspect, the present invention provides compositions for thecollection of stem cells, including pluripotent and multipotent stemcells, and committed progenitor cells, from organs, e.g., mammalianplacenta, either through physical disruption, e.g., enzymatic digestion,or by perfusion. The compositions facilitate collection of the stemcells, and inhibit apoptosis and/or necrosis of the stem cells,preferably during both collection and subsequent storage. Methods forcollecting stem cells, e.g., placental stem cells, using the stem cellcollection compositions are discussed below.

In one embodiment, the invention provides a composition for thecollection of stem cells from an organ, e.g., placental stem cells,comprising a physiologically-acceptable aqueous solution, and aninhibitor of apoptosis, wherein said composition, when contacted with apopulation of stem cells, reduces or prevents apoptosis in saidpopulation of stem cells as compared to a population of stem cells notcontacted with the composition. In one embodiment, the composition isnot a naturally-occurring composition. Preferably, thephysiologically-acceptable aqueous solution is suitable for themaintenance of a stem cell. The physiologically acceptable aqueoussolution can be, for example, an aqueous isotonic solution, such as aphysiologically-acceptable aqueous solution, e.g., a buffered salinesolution such as 0.9% NaCl solution, phosphate buffered saline,Dulbecco's Modified Eagle's Medium (DMEM), high-glucose Dulbecco'sModified Eagle's Medium (h.DMEM), Krebs solution, and the like. Thephysiologically-acceptable aqueous solution can be a culture medium,that is, any medium ordinarily used for the culture of mammalian cells,wherein the medium either includes or lacks, e.g., antibiotics or serum.In a specific embodiment, the physiologically acceptable aqueoussolution comprises sodium chloride, potassium chloride, magnesiumsulfate, calcium chloride, sodium sulfate, potassium sulfate, sodiumcarbonate, and glucose. The physiologically-acceptable aqueous solutioncan also comprise, or lack, any essential or non-essential amino acid,or combinations of amino acids.

The stem cell collection composition preferably comprises an enzymecapable of disrupting tissue, e.g., a protease, for example, a proteasedescribed in Section 5.1.1, below. The stem cell collection compositioncomprises an inhibitor of apoptosis, which can be any inhibitor ofapoptosis known in the art, e.g., a caspase inhibitor, e.g., a caspaseinhibitor described in Section 5.1.2.1, below. Preferably, the inhibitorof apoptosis is a JNK inhibitor, e.g., a JNK inhibitor described inSection 5.1.2.2, below. In another embodiment, thephysiologically-acceptable aqueous solution can also comprise animmunomodulatory compound, e.g., an immunomodulatory compound describedin Section 5.1.3, below. The physiologically-acceptable aqueous solutioncan also comprise a vasodilator, e.g., a vasodilator described inSection 5.1.4, below. The physiologically-acceptable aqueous solutioncan comprise an inhibitor of necrosis, e.g., an inhibitor of necrosisdescribed in Section 5.1.5, below. The physiologically-acceptablesolution can comprise an oxygen-carrying perfluorocarbon, e.g., aperfluorocarbon described in Section 5.1.6, below. Thephysiologically-acceptable solution can comprise one or a combination ofany of the foregoing compounds.

When used as a perfusion solution, the stem cell collection compositionof the invention preferably comprises an anticoagulant, such as heparin,citrate, citrate phosphate, citrate phosphate dextrose, CPDA (citratephosphate dextrose adenine), or the like, at a concentration sufficientto prevent the formation of clots of any residual cord or placentalblood. In a specific embodiment, from about 1 to about 100 units ofheparin is employed for the collection of stem cells from a singlemammalian placenta, and preferably about 1 to about 10 units of heparinper ml is employed.

5.1.1 Enzymes

The composition can comprise one or more enzymes that act to disrupttissue and/or the junctions between cells, or between cells and abasement membrane. In one embodiment, the enzyme is present in an amountsufficient to dissociate a detectable plurality of stem cells from anorgan or tissue, e.g., a placenta, from which stem cells may be derived.Such an enzyme can be, for example, a protease or a polypeptide havingprotease activity. The protease may be human, mammalian, bacterial,etc., and can be a native protease polypeptide, or a modifiedpolypeptide (e.g., sequence variant, truncation, fusion protein, analog)having protease activity. In various embodiments, the protease is amatrix metalloprotease or a neutral protease. In other embodiments, theenzyme is collagenase (e.g., collagenase I, collagenase IV, acollagenase from Clostridium histolyticum, etc.), trypsin (e.g.,trypsin-EDTA), thermolysin, elastase, dispase, LIBERASE™ or acombination thereof. Such enzymes may be obtained from commercialsources, e.g., SigmaAldrich (St. Louis, Miss.); Roche Diagnostics(LIBERASE™; Indianapolis, Ind.); Clinalfa (Bloomington, Ind.). Enzymescan be used in any effective concentration, e.g., about 0.1 mg/mL toabout 10 mg/mL. In various specific embodiments, trypsin-EDTA (GibcoBRL)can be used at a concentration of about 0.25% (w/v); collagenase-IA(Sigma) can be used at a concentration of about 1 mg/mL; collagenase-I(Worthington) can be used at a concentration of about 0.5 mg/mL;elastase can be used at a concentration of about 1 mg/ml; collagenase-IVcan be used at a concentration of about 0.5 mg/mL, and dispase(Worthington) can be used at a concentration of about 0.1 mg/mL.Preferred combinations of enzymes include collagenase I+trypsin;collagenase 1A+trypsin, and elastase+collagenase 1+collagenaseIV+dispase. Persons of skill in the art will understand that the exampleworking concentrations provided above can be increased or decreased tooptimize a particular digestion or perfusion protocol.

The composition can also comprise a nuclease, e.g., a DNase or RNase.The composition can additionally comprise a mucolytic enzyme. In aspecific embodiment, said mucolytic enzyme is hyaluronidase.

5.1.2 Apoptosis Inhibitors

The stem cell collection composition of the present invention comprisesan agent that reduces, suppresses or eliminates apoptosis of placentalcells, particularly the stem cells to be collected, e.g., an apoptosisinhibitor. Preferably, the agent reduces, suppresses or eliminatesapoptosis of placental stem cells during collection and duringsubsequent storage. The inhibitor of apoptosis may be any knownapoptosis inhibitor, e.g., a caspase inhibitor, but is preferably a JNKinhibitor.

5.1.2.1 Caspase Inhibitors

The stem cell collection composition of the invention can comprise acaspase inhibitor. The caspase inhibitor can be an inhibitor of aparticular caspase, e.g., caspase 1, caspase 2, caspase 3, caspase 4,caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10,caspase 11, caspase 12, or caspase 13. The caspase inhibitor can inhibitseveral caspases, or can inhibit all known caspases (e.g., pan-caspaseinhibitors). The caspase inhibitor can be an inhibitor of a caspaseprotein, or an inhibitor of activation of a caspase, e.g., inhibitor ofa gene encoding a caspase. Examples of caspase inhibitors include, butare not limited to, 2,2′-methylenebis(1,3-cyclohexanedione), a peptidehaving the sequence Z-Val-Ala-Asp(OMe)-CH₂F, a peptide having thesequence Biotin-X-Val-Ala-Asp(OMe)-CH₂F where X is a linker, a peptidehaving the sequence Ac-Val-Ala-Asp-CHO, Boc-Asp(OMe)-CH₂F, a peptidehaving the sequence Z-Val-Asp(OMe)-Val-Ala-Asp(OMe)-CH₂F, a peptidehaving the sequence Ac-Asp-Glu-Val-Asp-CHO, a peptide having thesequence Ac-Leu-Glu-Cal-Asp-CHO, a peptide having the sequenceZ-Trp-Glu(OMe)-His-Asp(OMe)-CH₂F, and the like. Caspase inhibitors maybe obtained from, e.g., CalBiochem (San Diego, Calif.), BioVision(Mountain View, Calif.); Clontech (Mountain View, Calif.), or R&DSystems, Inc. (Minneapolis, Minn.).

5.1.2.2 JNK Inhibitors

The apoptosis inhibitor of the stem cell collection composition ispreferably a JNK inhibitor; more preferably, the JNK inhibitor is acompound disclosed herein. Without wishing to be bound by theory, theaddition of an apoptosis inhibitor, particularly a JNK inhibitor,facilitates the collection of stem cells from mammalian placenta byincreasing the numbers of viable stem cells that may be collected, andmore closely maintaining the cellular environment surrounding such stemcells during the process of collection and isolation.

In a specific embodiment, the JNK inhibitor does not modulate thedifferentiation or proliferation of a stem cell or population of stemcells contacted with the stem cell collection composition comprising theJNK inhibitor; that is, the JNK inhibitor does not cause a detectabledifference in the proliferation or differentiation of the stem cell orstem cell population as compared to a stem cell not contacted with theJNK inhibitor.

JNK inhibitors used in the compositions and methods of the inventioninclude racemic, stereomerically pure and stereomerically enriched JNKinhibitors, stereomerically and enantiomerically pure compounds thathave selective JNK inhibitory activities, and pharmaceuticallyacceptable salts, solvates, hydrates, stereoisomers, clathrates, andprodrugs thereof. Such JNK inhibitors can either be commerciallypurchased or prepared according to the methods described in the patentsor patent publications disclosed herein. Further, optically purecompositions can be asymmetrically synthesized or resolved using knownresolving agents or chiral columns as well as other standard syntheticorganic chemistry techniques.

In one embodiment, a JNK inhibitor has the following structure (I):

wherein:

A is a direct bond, —(CH₂)_(a)—, —(CH₂)_(b)CH═CH(CH₂)C—, or—(CH₂)_(b)C≡C(CH₂)_(c)—;

R₁ is aryl, heteroaryl or heterocycle fused to phenyl, each beingoptionally substituted with one to four substituents independentlyselected from R₃;

R₂ is —R₃, —R₄, —(CH₂)_(b)C(═O)R₅, —(CH₂)_(b)C(═O)OR₅,—(CH₂)_(b)C(═O)NR₅R₆,

—(CH₂)_(b)C(═O)NR₅(CH₂)_(c)C(═O)R₆, —(CH₂)_(b)NR₅C(═O)R₆,

—(CH₂)_(b)NR₅C(═O)NR₆R₇, —(CH₂)_(b)NR₅R₆, —(CH₂)_(b)OR₅,

—(CH₂)_(b)SO_(d)R₅ or —(CH₂)_(b)SO₂NR₅R₆;

a is 1, 2, 3, 4, 5 or 6;

b and c are the same or different and at each occurrence independentlyselected from 0, 1, 2, 3 or 4;

d is at each occurrence 0, 1 or 2;

R₃ is at each occurrence independently halogen, hydroxy, carboxy, alkyl,alkoxy, haloalkyl, acyloxy, thioalkyl, sulfinylalkyl, sulfonylalkyl,hydroxyalkyl, aryl, arylalkyl, heterocycle, heterocycloalkyl, —C(═O)OR₈,—OC(═O)R₈, —C(═O)NR₈R₉, —C(═O)NR₈OR₉, —SO₂NR₉R₉, —NR₈SO₂R₉, —CN, —NO₂,—NR₉R₉, —NR₉C(═O)R₉, —NR₉C(═O)(CH₂)_(b)OR₉, —NR₈C(═O)(CH₂)_(b)R₉,—O(CH₂)_(b)NR₈R₉, or heterocycle fused to phenyl;

R₄ is alkyl, aryl, arylalkyl, heterocycle or heterocycloalkyl, eachbeing optionally substituted with one to four substituents independentlyselected from R₃, or R₄ is halogen or hydroxy;

R₅, R₆ and R₇ are the same or different and at each occurrenceindependently hydrogen, alkyl, aryl, arylalkyl, heterocycle orheterocycloalkyl, wherein each of R₅, R₆ and

R₇ are optionally substituted with one to four substituentsindependently selected from R₃; and

R₈ and R₉ are the same or different and at each occurrence independentlyhydrogen, alkyl, aryl, arylalkyl, heterocycle, or heterocycloalkyl, orR₈ and R₉ taken together with the atom or atoms to which they are bondedform a heterocycle, wherein each of R₈, R₉, and R₉ and R₉ taken togetherto form a heterocycle are optionally substituted with one to foursubstituents independently selected from R₃.

In one embodiment, -A-R₁ is phenyl, optionally substituted with one tofour substituents independently selected from halogen, alkoxy,—NR8C(═O)R9, —C(═O)NR8R₉, and —O(CH2)_(b)NR8R9, wherein b is 2 or 3 andwherein R8 and R9 are defined above.

In another embodiment, R2 is —R4, —(CH2)_(b)C(═O)R5, —(CH2)_(b)C(═O)OR5,—(CH2)_(b)C(═O)NR5R6, —(CH2)_(b)C(═O)NR5(CH2)_(c)C(═O)R6,—(CH2)_(b)NR5C(═O)R6, —(CH2)_(b)NR5C(═O)NR6R7, —(CH2)_(b)NR5R6,—(CH2)_(b)OR5, —(CH2)_(b)SOdR5 or —(CH2)_(b)SO2NR5R6, and b is aninteger ranging from 0-4.

In another embodiment, R2 is —(CH2)_(b)C(═O)NR5R6, —(CH2)_(b)NR5C(═O)R6,3-triazolyl or 5-tetrazolyl, wherein b is 0 and wherein R8 and R9 aredefined above.

In another embodiment, R2 is 3-triazolyl or 5-tetrazolyl.

In another embodiment:

(a) -A-R₁ is phenyl, optionally substituted with one to foursubstituents independently selected from halogen, alkoxy, —NR₈C(═O)R₉,—C(═O)NR₈R₉,

and —O(CH₂)_(b)NR₈R₉, wherein b is 2 or 3; and

(b) R₂ is —(CH₂)_(b)C(═O)NR₅R₆, —(CH₂)_(b)NR₅C(═O)R₆, 3-triazolyl or5-tetrazolyl, wherein b is 0 and wherein R₈ and R₉ are defined above.

In another embodiment:

(a) -A-R₁ is phenyl, optionally substituted with one to foursubstituents independently selected from halogen, alkoxy, —NR₈C(═O)R₉,—C(═O)NR₈R₉, and —O(CH₂)_(b)NR₈R₉, wherein b is 2 or 3; and

(b) R₂ is 3-triazolyl or 5-tetrazolyl.

In another embodiment, R₂ is R₄, and R₄ is 3-triazolyl, optionallysubstituted at its 5-position with:

(a) a C₁-C₄ straight or branched chain alkyl group optionallysubstituted with a hydroxyl, methylamino, dimethylamino or1-pyrrolidinyl group; or

(b) a 2-pyrrolidinyl group.

In another embodiment, R₂ is R₄, and R₄ is 3-triazolyl, optionallysubstituted at its 5-position with: methyl, n-propyl, isopropyl,1-hydroxyethyl, 3-hydroxypropyl, methylaminomethyl, dimethylaminomethyl,1-(dimethylamino)ethyl, 1-pyrrolidinylmethyl or 2-pyrrolidinyl.

In another embodiment, the compounds of structure (I) have structure(IA) when A is a direct bond, or have structure (IB) when A is—(CH₂)_(a)—:

In other embodiments, the compounds of structure (I) have structure (IC)when A is a —(CH₂)_(b)CH═CH(CH₂)C—, and have structure (ID) when A is—(CH₂)_(b)C≡C(CH₂)_(c)—:

In further embodiments of this invention, R₁ of structure (I) is aryl orsubstituted aryl, such as phenyl or substituted phenyl as represented bythe following structure (IE):

In another embodiment, R₂ of structure (I) is —(CH₂)_(b)NR₄(C═O)R₅. Inone aspect of this embodiment, b=0 and the compounds have the followingstructure (IF):

Representative R₂ groups of the compounds of structure (I) include alkyl(such as methyl and ethyl), halo (such as chloro and fluoro), haloalkyl(such as trifluoromethyl), hydroxy, alkoxy (such as methoxy and ethoxy),amino, arylalkyloxy (such as benzyloxy), mono- or di-alkylamine (such as—NHCH₃, —N(CH₃)₂ and —NHCH₂CH₃), —NHC(═O)R₄ wherein R₆ is a substitutedor unsubstituted phenyl or heteroaryl (such as phenyl or heteroarylsubstituted with hydroxy, carboxy, amino, ester, alkoxy, alkyl, aryl,haloalkyl, halo, —CONH₂ and —CONH alkyl), —NH(heteroarylalkyl) (such as—NHCH₂(3-pyridyl), —NHCH₂(4-pyridyl), heteroaryl (such as pyrazolo,triazolo and tetrazolo), —C(═O)NHR₆ wherein R₆ is hydrogen, alkyl, or asdefined above (such as —C(═O)NH₂, —C(═O)NHCH₃,—C(═O)NH(H-carboxyphenyl), —C(═O)N(CH₃)₂), arylalkenyl (such asphenylvinyl, 3-nitrophenylvinyl, 4-carboxyphenylvinyl),heteroarylalkenyl (such as 2-pyridylvinyl, 4-pyridylvinyl).

Representative R₃ groups of the compounds of structure (I) includehalogen (such as chloro and fluoro), alkyl (such as methyl, ethyl andisopropyl), haloalkyl (such as trifluoromethyl), hydroxy, alkoxy (suchas methoxy, ethoxy, n-propyloxy and isobutyloxy), amino, mono- ordi-alkylamino (such as dimethylamine), aryl (such as phenyl), carboxy,nitro, cyano, sulfinylalkyl (such as methylsulfinyl), sulfonylalkyl(such as methylsulfonyl), sulfonamidoalkyl (such as —NHSO₂CH₃),—NR₈C(═O)(CH₂)_(b)OR₉ (such as NHC(═O)CH₂OCH₃), NHC(═O)R₉ (such as—NHC(═O)CH₃, —NHC(═O)CH₂C₆H₅, —NHC(═O)(2-furanyl)), and —O(CH₂)_(b)NR₉R₉(such as —O(CH₂)₂N(CH₃)₂).

The compounds of structure (I) can be made using organic synthesistechniques known to those skilled in the art, as well as by the methodsdescribed in International Publication No. WO 02/10137 (particularly inExamples 1-430, at page 35, line 1 to page 396, line 12), published Feb.7, 2002, which is incorporated herein by reference in its entirety.Further, specific examples of these compounds are found in thispublication.

Illustrative examples of JNK inhibitors of structure (I) are:

and pharmaceutically acceptable salts thereof.

In another embodiment, the JNK inhibitor has the following structure(II):

wherein:

R₁ is aryl or heteroaryl optionally substituted with one to foursubstituents independently selected from R₇;

R₂ is hydrogen;

R₃ is hydrogen or lower alkyl;

R₄ represents one to four optional substituents, wherein eachsubstituent is the same or different and independently selected fromhalogen, hydroxy, lower alkyl and lower alkoxy;

R₅ and R₆ are the same or different and independently —R₈,—(CH₂)_(a)C(═O)R₉, —(CH₂)_(a)C(═O)OR₉, —(CH₂)_(a)C(═O)NR₉R₁₀,—(CH₂)_(a)C(═O)NR₉(CH₂)_(b)C(═O)R₁₀, —(CH₂)_(a)NR₉C(═O)R₁₀,(CH₂)_(a)NR₁₁C(═O)NR₉R₁₀, —(CH₂)_(a)NR₉R₁₀, —(CH₂)_(a)OR₉,—(CH₂)_(a)SO_(c)R₉ or —(CH₂)_(a)SO₂NR₉R₁₀;

or R₅ and R₆ taken together with the nitrogen atom to which they areattached to form a heterocycle or substituted heterocycle;

R₇ is at each occurrence independently halogen, hydroxy, cyano, nitro,carboxy, alkyl, alkoxy, haloalkyl, acyloxy, thioalkyl, sulfinylalkyl,sulfonylalkyl, hydroxyalkyl, aryl, arylalkyl, heterocycle, substitutedheterocycle, heterocycloalkyl, —C(═O)OR₈, —OC(═O)R₈, —C(═O)NR₈R₉,—C(═O)NR₈OR₉, —SO_(c)R₈, —SO_(c)NR₈R₉, —NR₈SO_(c)R₉, —NR₈R₉,—NR₈C(═O)R₉, —NR₈C(═O)(CH₂)_(b)OR₉, —NR₈C(═O)(CH₂)_(b)R₉,—O(CH₂)_(b)NR₈R₉, or heterocycle fused to phenyl;

R₈, R₉, R₁₀ and R₁₁ are the same or different and at each occurrenceindependently hydrogen, alkyl, aryl, arylalkyl, heterocycle,heterocycloalkyl;

or R₈ and R₉ taken together with the atom or atoms to which they areattached to form a heterocycle;

a and b are the same or different and at each occurrence independentlyselected from 0, 1, 2, 3 or 4; and

c is at each occurrence 0, 1 or 2.

In one embodiment, R₁ is a substituted or unsubstituted aryl orheteroaryl. When R₁ is substituted, it is substituted with one or moresubstituents defined below. In one embodiment, when substituted, R₁ issubstituted with a halogen, —SO₂R₈ or —SO₂R₈R₉.

In another embodiment, R₁ is substituted or unsubstituted aryl, furyl,benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl,indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl,benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl orquinazolinyl.

In another embodiment R₁ is substituted or unsubstituted aryl orheteroaryl. When R₁ is substituted, it is substituted with one or moresubstituents defined below. In one embodiment, when substituted, R₁ issubstituted with a halogen, —SO₂R₈ or —SO₂R₈R₉.

In another embodiment, R₁ is substituted or unsubstituted aryl,preferably phenyl. When R₁ is a substituted aryl, the substituents aredefined below. In one embodiment, when substituted, R₁ is substitutedwith a halogen, —SO₂R₈ or —SO₂R₈R₉.

In another embodiment, R₅ and R₆, taken together with the nitrogen atomto which they are attached form a substituted or unsubstitutednitrogen-containing non-aromatic heterocycle, in one embodiment,piperazinyl, piperidinyl or morpholinyl.

When R₅ and R₆, taken together with the nitrogen atom to which they areattached form substituted piperazinyl, piperadinyl or morpholinyl, thepiperazinyl, piperadinyl or morpholinyl is substituted with one or moresubstituents defined below. In one embodiment, when substituted, thesubstituent is alkyl, amino, alkylamino, alkoxyalkyl, acyl, pyrrolidinylor piperidinyl.

In one embodiment, R₃ is hydrogen and R₄ is not present, and the JNKinhibitor has the following structure (IIA):

and pharmaceutically acceptable salts thereof.

In a more specific embodiment, R₁ is phenyl optionally substituted withR₇, and having the following structure (IIB):

and pharmaceutically acceptable salts thereof.

In still a further embodiment, R₇ is at the para position of the phenylgroup relative to the pyrimidine, as represented by the followingstructure (IIC):

and pharmaceutically acceptable salts thereof.

The JNK inhibitors of structure (II) can be made using organic synthesistechniques known to those skilled in the art, as well as by the methodsdescribed in International Publication No. WO 02/46170 (particularlyExamples 1-27 at page 23, line 5 to page 183, line 25), published Jun.13, 2002, which is hereby incorporated by reference in its entirety.Further, specific examples of these compounds are found in thepublication.

Illustrative examples of JNK inhibitors of structure (II) are:

and pharmaceutically acceptable salts thereof.

In another embodiment, the JNK inhibitor has the following structure(III):

wherein R₀ is —O—, —S—, —S(O)—, —S(O)₂—, NH or —CH₂—;

the compound of structure (III) being: (i) unsubstituted, (ii)monosubstituted and having a first substituent, or (iii) disubstitutedand having a first substituent and a second substituent;

the first or second substituent, when present, is at the 3, 4, 5, 7, 8,9, or 10 position, wherein the first and second substituent, whenpresent, are independently alkyl, hydroxy, halogen, nitro,trifluoromethyl, sulfonyl, carboxyl, alkoxycarbonyl, alkoxy, aryl,aryloxy, arylalkyloxy, arylalkyl, cycloalkylalkyloxy, cycloalkyloxy,alkoxyalkyl, alkoxyalkoxy, aminoalkoxy, mono-alkylaminoalkoxy,di-alkylaminoalkoxy, or a group represented by structure (a), (b), (c),(d), (e), or (f):

wherein R₃ and R₄ are taken together and represent alkylidene or aheteroatom-containing cyclic alkylidene or R₃ and R₄ are independentlyhydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,aryloxyalkyl, alkoxyalkyl, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonylalkyl, amino, mono-alkylamino,di-alkylamino, arylamino, arylalkylamino, cycloalkylamino,cycloalkylalkylamino, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl.

In another embodiment, the JNK inhibitor has the following structure(IIIA):

being: (i) unsubstituted, (ii) monosubstituted and having a firstsubstituent, or (iii) disubstituted and having a first substituent and asecond substituent;

the first or second substituent, when present, is at the 3, 4, 5, 7, 8,9, or 10 position;

wherein the first and second substituent, when present, areindependently alkyl, hydroxy, halogen, nitro, trifluoromethyl, sulfonyl,carboxyl, alkoxycarbonyl, alkoxy, aryl, aryloxy, arylalkyloxy,arylalkyl, cycloalkylalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy,aminoalkoxy, mono-alkylaminoalkoxy, di-alkylaminoalkoxy, or a grouprepresented by structure (a), (b), (c), (d), (e), or (f):

wherein R₃ and R₄ are taken together and represent alkylidene or aheteroatom-containing cyclic alkylidene or R₃ and R₄ are independentlyhydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,aryloxyalkyl, alkoxyalkyl, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonylalkyl, amino, mono-alkylamino,di-alkylamino, arylamino, arylalkylamino, cycloalkylamino,cycloalkylalkylamino, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl.

A subclass of the compounds of structure (IIIA) is that wherein thefirst or second substituent is present at the 5, 7, or 9 position. Inone embodiment, the first or second substituent is present at the 5 or 7position.

A second subclass of compounds of structure (IIIA) is that wherein thefirst or second substituent is present at the 5, 7, or 9 position;

the first or second substituent is independently alkoxy, aryloxy,aminoalkyl, mono-alkylaminoalkyl, di-alkylaminoalkyl, or a grouprepresented by the structure (a), (c), (d), (e), or (f);

R₃ and R₄ are independently hydrogen, alkyl, cycloalkyl, aryl,arylalkyl, or cycloalkylalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, or cycloalkylalkyl.

In another embodiment, the JNK inhibitor has the following structure(IIIB):

being (i) unsubstituted, (ii) monosubstituted and having a firstsubstituent, or (ii) disubstituted and having a first substituent and asecond substituent;

the first or second substituent, when present, is at the 3, 4, 5, 7, 8,9, or 10 position;

wherein the first and second substituent, when present, areindependently alkyl, halogen, hydroxy, nitro, trifluoromethyl, sulfonyl,carboxyl, alkoxycarbonyl, alkoxy, aryl, aryloxy, arylalkyloxy,arylalkyl, cycloalkylalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy,aminoalkoxy, mono-alkylaminoalkoxy, di-alkylaminoalkoxy, or a grouprepresented by structure (a), (b) (c), (d), (e), or (f):

wherein R₃ and R₄ are taken together and represent alkylidene or aheteroatom-containing cyclic alkylidene or R₃ and R₄ are independentlyhydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,aryloxyalkyl, alkoxyalkyl, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonylalkyl, amino, mono-alkylamino,di-alkylamino, arylamino, arylalkylamino, cycloalkylamino,cycloalkylalkylamino, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl.

A subclass of the compounds of structure (IIIB) is that wherein thefirst or second substituent is present at the 5, 7, or 9 position. Inone embodiment, the first or second substituent is present at the 5 or 7position.

A second subclass of the compounds of structure (IIIB) is that whereinthe first or second substituent is independently alkoxy, aryloxy, or agroup represented by the structure (a), (c), (d), (e), or (f);

R₃ and R₄ are independently hydrogen, alkyl, cycloalkyl, aryl,arylalkyl, or cycloalkylalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, or cycloalkylalkyl.

In another embodiment, the JNK inhibitor has the following structure(IIIC):

being (i) monosubstituted and having a first substituent or (ii)disubstituted and having a first substituent and a second substituent;

the first or second substituent, when present, is at the 3, 4, 5, 7, 8,9, or 10 position;

wherein the first and second substituent, when present, areindependently alkyl, halogen, hydroxy, nitro, trifluoromethyl, sulfonyl,carboxyl, alkoxycarbonyl, alkoxy, aryl, aryloxy, arylalkyloxy,arylalkyl, cycloalkylalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy,aminoalkoxy, mono-alkylaminoalkoxy, di-alkylaminoalkoxy, or a grouprepresented by structure (a), (b), (c) (d), (e), or (f):

wherein R₃ and R₄ are taken together and represent alkylidene or aheteroatom-containing cyclic alkylidene or R₃ and R₄ are independentlyhydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,aryloxyalkyl, alkoxyalkyl, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonylalkyl, amino, mono-alkylamino,di-alkylamino, arylamino, arylalkylamino, cycloalkylamino,cycloalkylalkylamino, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl.

A subclass of the compounds of structure (IIIC) is that wherein thefirst or second substituent is present at the 5, 7, or 9 position. Inone embodiment, the first or second substituent is present at the 5 or 7position.

A second subclass of the compounds of structure (IIIC) is that whereinthe first or second substituent is independently alkoxy, aryloxy,aminoalkyl, mono-alkylaminoalkyl, di-alkylaminoalkyl, or a grouprepresented by the structure (a), (c), (d), (e), or (f);

R₃ and R₄ are independently hydrogen, alkyl, cycloalkyl, aryl,arylalkyl, or cycloalkylalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, or cycloalkylalkyl.

In another embodiment, the JNK inhibitor has the following structure(IIID):

being (i) monosubstituted and having a first substituent present at the5, 7, or 9 position, (ii) disubstituted and having a first substituentpresent at the 5 position and a second substituent present at the 7position, (iii) disubstituted and having a first substituent present atthe 5 position and a second substituent present at the 9 position, or(iv) disubstituted and having a first substituent present at the 7position and a second substituent present at the 9 position;

wherein the first and second substituent, when present, areindependently alkyl, halogen, hydroxy, nitro, trifluoromethyl, sulfonyl,carboxyl, alkoxycarbonyl, alkoxy, aryl, aryloxy, arylalkyloxy,arylalkyl, cycloalkylalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy,aminoalkoxy, mono-alkylaminoalkoxy, di-alkylaminoalkoxy, or a grouprepresented by structure (a), (b), (c), (d), (e), or (f):

wherein R₃ and R₄ are taken together and represent alkylidene or aheteroatom-containing cyclic alkylidene or R₃ and R₄ are independentlyhydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,aryloxyalkyl, alkoxyalkyl, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonylalkyl, amino, mono-alkylamino,di-alkylamino, arylamino, arylalkylamino, cycloalkylamino,cycloalkylalkylamino, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl.

A subclass of the compounds of structure (IIID) is that wherein thefirst or second substituent is present at the 5 or 7 position.

A second subclass of the compounds of structure (IIID) is that whereinthe first or second substituent is independently alkyl, trifluoromethyl,sulfonyl, carboxyl, alkoxycarbonyl, alkoxy, aryl, aryloxy, arylalkyloxy,arylalkyl, cycloalkylalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy,aminoalkoxy, mono-alkylaminoalkoxy, di-alkylaminoalkoxy, or a grouprepresented by structure (a), (c), (d), (e), or (f).

Another subclass of the compounds of structure (IIID) is that whereinthe first and second substituent are independently alkoxy, aryloxy, or agroup represented by the structure (a), (c), (d), (e), or (f);

R₃ and R₄ are independently hydrogen, alkyl, cycloalkyl, aryl,arylalkyl, or cycloalkylalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, alkoxycarbonyl, orcycloalkylalkyl.

In another embodiment, the JNK inhibitor has the following structure(IIIE):

being (i) monosubstituted and having a first substituent present at the5, 7, or 9 position, (ii) disubstituted and having a first substituentpresent at the 5 position and a second substituent present at the 9position, (iii) disubstituted and having a first substituent present atthe 7 position and a second substituent present at the 9 position, or(iv) disubstituted and having a first substituent present at the 5position and a second substituent present at the 7 position;

wherein the first and second substituent, when present, areindependently alkyl, halogen, hydroxy, nitro, trifluoromethyl, sulfonyl,carboxyl, alkoxycarbonyl, alkoxy, aryl, aryloxy, arylalkyloxy,arylalkyl, cycloalkylalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy,aminoalkoxy, mono-alkylaminoalkoxy, di-alkylaminoalkoxy, or a grouprepresented by structure (a), (b), (c), (d), (e), or (f):

wherein R₃ and R₄ are taken together and represent alkylidene or aheteroatom-containing cyclic alkylidene or R₃ and R₄ are independentlyhydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,aryloxyalkyl, alkoxyalkyl, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonylalkyl, amino, mono-alkylamino,di-alkylamino, arylamino, arylalkylamino, cycloalkylamino,cycloalkylalkylamino, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl.

A subclass of the compounds of structure (IIIE) is that wherein thefirst or second substituent is present at the 5 or 7 position.

A second subclass of the compounds of structure (IIIE) is that whereinthe compound of structure (IIIE) is disubstituted and at least one ofthe substituents is a group represented by the structure (d) or (f).

Another subclass of the compounds of structure (IIIE) is that whereinthe compounds are monosubstituted. Yet another subclass of compounds isthat wherein the compounds are monosubstituted at the 5 or 7 positionwith a group represented by the structure (e) or (f).

In another embodiment, the JNK inhibitor has the following structure(IIIF):

being (i) unsubstituted, (ii) monosubstituted and having a firstsubstituent, or (iii) disubstituted and having a first substituent and asecond substituent;

the first or second substituent, when present, is at the 3, 4, 5, 7, 8,9, or 10 position;

wherein the first and second substituent, when present, areindependently alkyl, hydroxy, halogen, nitro, trifluoromethyl, sulfonyl,carboxyl, alkoxycarbonyl, alkoxy, aryl, aryloxy, arylalkyloxy,arylalkyl, cycloalkylalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy,aminoalkoxy, mono-alkylaminoalkoxy, di-alkylaminoalkoxy, or a grouprepresented by structure (a), (b), (c), (d), (e), or (f):

wherein R₃ and R₄ are taken together and represent alkylidene or aheteroatom-containing cyclic alkylidene or R₃ and R₄ are independentlyhydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,aryloxyalkyl, alkoxyalkyl, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl; and

R₅ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,alkoxy, alkoxyalkyl, alkoxycarbonylalkyl, amino, mono-alkylamino,di-alkylamino, arylamino, arylalkylamino, cycloalkylamino,cycloalkylalkylamino, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl.

In one embodiment, the compound of structure (IIIF), or apharmaceutically acceptable salt thereof is unsubstituted at the 3, 4,5, 7, 8, 9, or 10 position.

The JNK inhibitors of structure (III) can be made using organicsynthesis techniques known to those skilled in the art, as well as bythe methods described in International Publication No. WO 01/12609(particularly Examples 1-7 at page 24, line 6 to page 49, line 16),published Feb. 22, 2001, as well as International Publication No. WO02/066450 (particularly compounds AA-HG at pages 59-108), published Aug.29, 2002, each of which is hereby incorporated by reference in itsentirety. Further, specific examples of these compounds can be found inthe publications.

Illustrative examples of JNK inhibitors of structure (III) are:

and pharmaceutically acceptable salts thereof.

Other JNK inhibitors that are useful in the present compositions andmethods include, but are not limited to, those disclosed inInternational Publication No. WO 00/39101, (particularly at page 2, line10 to page 6, line 12); International Publication No. WO 01/14375(particularly at page 2, line 4 to page 4, line 4); InternationalPublication No. WO 00/56738 (particularly at page 3, line 25 to page 6,line 13); International Publication No. WO 01/27089 (particularly atpage 3, line 7 to page 5, line 29); International Publication No. WO00/12468 (particularly at page 2, line 10 to page 4, line 14); EuropeanPatent Publication 1 110 957 (particularly at page 19, line 52 to page21, line 9); International Publication No. WO 00/75118 (particularly atpage 8, line 10 to page 11, line 26); International Publication No. WO01/12621 (particularly at page 8, line 10 to page 10, line 7);International Publication No. WO 00/64872 (particularly at page 9, line1 to page, 106, line 2); International Publication No. WO 01/23378(particularly at page 90, line 1 to page 91, line 11); InternationalPublication No. WO 02/16359 (particularly at page 163, line 1 to page164, line 25); U.S. Pat. No. 6,288,089 (particularly at column 22, line25 to column 25, line 35); U.S. Pat. No. 6,307,056 (particularly atcolumn 63, line 29 to column 66, line 12); International Publication No.WO 00/35921 (particularly at page 23, line 5 to page 26, line 14);International Publication No. WO 01/91749 (particularly at page 29,lines 1-22); International Publication No. WO 01/56993 (particularly inat page 43 to page 45); and International Publication No. WO 01/58448(particularly in at page 39), each of which is incorporated by referenceherein in its entirety.

The stem cell collection composition of the invention may comprise oneor more solvents or co-solvents to facilitate solvation of a JNKinhibitor. Examples of solvents or co-solvents that can be included inthe stem cell collection composition include, but are not limited to,dimethylsulfoxide, ethanol, dimethylformamide, ethylene glycol,propylene glycol, and polyethylene glycol. Generally, only as muchsolvent or co-solvent is used as necessary to achieve a particularconcentration of a particular JNK inhibitor. Preferably, only solventsor co-solvents, or concentrations of solvents or co-solvents, that arecompatible with stem cell collection and culture are used.

It should be noted that if there is a discrepancy between a depictedstructure and a name given that structure, the depicted structure is tobe accorded more weight. In addition, if the stereochemistry of astructure or a portion of a structure is not indicated with, forexample, bold or dashed lines, the structure or portion of the structureis to be interpreted as encompassing all stereoisomers of it.

5.1.2.3 Other Apoptosis Inhibitors

Any other inhibitor of apoptosis can be included within the stem cellcomposition of the invention. For example, in various embodiments, theapoptosis inhibitor can be 2,2′-methylenebis(1,3-cyclohexanedione)(CalBiochem); apoptosis inhibitor 3 protein (i.e., XIAP, Baculoviral IAPrepeat containing protein 4, AP13, Mammalian IAP homolog A, MIHA,Inhibitor of apoptosis protein 3, X-linked inhibitor of apoptosisprotein, X-linked IAP, HILP, IAP Like protein. ILP); protein Mcl-1; andthe like.

5.1.3 TNF Alpha Inhibitors/Immunomodulatory Compounds

The composition of the invention can include one or more inhibitors ofTNF-α, e.g., an immunomodulatory compound. The immunomodulatory compoundcan be, e.g., thalidomide or a thalidomide derivative.

Specific examples of immunomodulatory compounds, include, but are notlimited to, cyano and carboxy derivatives of substituted styrenes suchas those disclosed in U.S. Pat. No. 5,929,117;1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3yl) isoindolines and1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl) isoindolines such asthose described in U.S. Pat. Nos. 5,874,448 and 5,955,476; the tetrasubstituted 2-(2,6-dioxopiperdin-3-yl)-1-oxoisoindolines described inU.S. Pat. No. 5,798,368; 1-oxo and1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindolines (e.g., 4-methylderivatives of thalidomide), including, but not limited to, thosedisclosed in U.S. Pat. Nos. 5,635,517, 6,476,052, 6,555,554, and6,403,613; 1-oxo and 1,3-dioxoisoindolines substituted in the 4- or5-position of the indoline ring (e.g.,4-(4-amino-1,3-dioxoisoindoline-2-yl)-4-carbamoylbutanoic acid)described in U.S. Pat. No. 6,380,239; isoindoline-1-one andisoindoline-1,3-dione substituted in the 2-position with2,6-dioxo-3-hydroxypiperidin-5-yl (e.g.,2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-aminoisoindolin-1-one)described in U.S. Pat. No. 6,458,810; a class of non-polypeptide cyclicamides disclosed in U.S. Pat. Nos. 5,698,579 and 5,877,200;aminothalidomide, as well as analogs, hydrolysis products, metabolites,derivatives and precursors of aminothalidomide, and substituted2-(2,6-dioxopiperidin-3-yl)phthalimides and substituted2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles such as those described inU.S. Pat. Nos. 6,281,230 and 6,316,471; and isoindole-imide compoundssuch as those described in U.S. patent application Ser. No. 09/972,487filed on Oct. 5, 2001, U.S. patent application Ser. No. 10/032,286 filedon Dec. 21, 2001, and International Application No. PCT/US01/50401(International Publication No. WO 02/059106). The entireties of each ofthe patents and patent applications identified herein are incorporatedherein by reference. Immunomodulatory compounds do not includethalidomide.

Other specific immunomodulatory compounds include, but are not limitedto, 1-oxo- and 1,3 dioxo-2-(2,6-dioxopiperidin-3-yl)isoindolinessubstituted with amino in the benzo ring as described in U.S. Pat. No.5,635,517 which is incorporated herein by reference. These compoundshave the structure I:

in which one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl, in particular methyl. Specificimmunomodulatory compounds include, but are not limited to:

-   1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline;-   1-oxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline;-   1-oxo-2-(2,6-dioxopiperidin-3-yl)-6-aminoisoindoline;-   1-oxo-2-(2,6-dioxopiperidin-3-yl)-7-aminoisoindoline;-   1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline; and-   1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline.

Other specific immunomodulatory compounds of the invention belong to aclass of substituted 2-(2,6-dioxopiperidin-3-yl)phthalimides andsubstituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles, such as thosedescribed in U.S. Pat. Nos. 6,281,230; 6,316,471; 6,335,349; and6,476,052, and International Patent Application No. PCT/US97/13375(International Publication No. WO 98/03502), each of which isincorporated herein by reference. Representative compounds are offormula:

in which:

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

(i) each of R¹, R², R³, and R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, and R⁴ is —NHR⁵ and the remaining of R¹, R², R³, andR⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbon atoms;

R⁶ is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl, or halo;

provided that R⁶ is other than hydrogen if X and Y are C═O and (i) eachof R¹, R², R³, and R⁴ is fluoro or (ii) one of R¹, R², R³, or R⁴ isamino.

Compounds representative of this class are of the formulas:

wherein R¹ is hydrogen or methyl. In a separate embodiment, theinvention encompasses the use of enantiomerically pure forms (e.g.optically pure (R) or (S) enantiomers) of these compounds.

Still other specific immunomodulatory compounds of the invention belongto a class of isoindole-imides disclosed in U.S. Patent ApplicationPublication Nos. US 2003/0096841 and US 2003/0045552, and InternationalApplication No. PCT/US01/50401 (International Publication No. WO02/059106), each of which are incorporated herein by reference.Representative compounds are of formula II:

and pharmaceutically acceptable salts, hydrates, solvates, clathrates,enantiomers, diastereomers, racemates, and mixtures of stereoisomersthereof, wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R¹ is H, (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂₋₈)alkenyl, (C₂-C₈)alkynyl,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴,(C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³,C(S)NHR³, C(O)NR³R^(3′), C(S)NR³R^(3′) or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl;

R³ and R^(3′) are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl,(C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵,benzyl, aryl, (C₀-C₄)alkyl-C₁-C₆)heterocycloalkyl, or(C₀-C₄)alkyl-C₂-C₅)heteroaryl;

R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, or(C₂-C₅)heteroaryl;

each occurrence of R⁶ is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₂-C₅)heteroaryl, or(C₀-C₈)alkyl-C(O)O—R⁵ or the R⁶ groups can join to form aheterocycloalkyl group;

n is 0 or 1; and

* represents a chiral-carbon center.

In specific compounds of formula II, when n is 0 then R¹ is(C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl,C(O)R³, C(O)OR⁴, (C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵,(C₁-C₈)alkyl-C(O)OR⁵, C(S)NHR³, or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H or (C₁-C₈)alkyl; and

R³ is (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, (C₅-C₈)alkyl-N(R⁶)₂;(C₀-C₈)alkyl-NH—C(O)O—R⁵; (C₁-C₈)alkyl-OR₅, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵; and the other variables have the samedefinitions.

In other specific compounds of formula II, R² is H or (C₁-C₄)alkyl.

In other specific compounds of formula II, R¹ is (C₁-C₈)alkyl or benzyl.

In other specific compounds of formula II, R¹ is H, (C₁-C₈)alkyl,benzyl, CH₂OCH₃, CH₂CH₂OCH₃, or

In another embodiment of the compounds of formula II, R¹ is

wherein Q is O or S, and each occurrence of R⁷ is independentlyH,(C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,benzyl, aryl, halogen, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₈)heteroaryl, (C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵,(C₁-C₈)alkyl-C(O)OR⁵, (C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵, or adjacentoccurrences of R⁷ can be taken together to form a bicyclic alkyl or arylring.

In other specific compounds of formula II, R¹ is C(O)R³.

In other specific compounds of formula II, R³ is(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, (C₁-C₈)alkyl, aryl, or (C₀-C₄)alkyl-OR⁵.

In other specific compounds of formula II, heteroaryl is pyridyl, furyl,or thienyl.

In other specific compounds of formula II, R¹ is C(O)OR⁴.

In other specific compounds of formula II, the H of C(O)NHC(O) can bereplaced with (C₁-C₄)alkyl, aryl, or benzyl.

Further examples of the compounds in this class include, but are notlimited to:[2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl]-amide;(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl)-carbamicacid tert-butyl ester;4-(aminomethyl)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione;N-(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl)-acetamide;N-{(2-(2,6-dioxo(3-piperidyl)-1,3-dioxoisoindolin-4-yl)methyl}cyclopropyl-carboxamide;2-chloro-N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}acetamide;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-3-pyridylcarboxamide;3-{1-oxo-4-(benzylamino)isoindolin-2-yl}piperidine-2,6-dione;2-(2,6-dioxo(3-piperidyl))-4-(benzylamino)isoindoline-1,3-dione;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}propanamide;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-3-pyridylcarboxamide;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}heptanamide;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-2-furylcarboxamide;{N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)carbamoyl}methylacetate;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)pentanamide;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-2-thienylcarboxamide;N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(butylamino)carboxamide;N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(octylamino)carboxamide;andN-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(benzylamino)carboxamide.

Still other specific immunomodulatory compounds of the invention belongto a class of isoindole-imides disclosed in U.S. Patent ApplicationPublication Nos. US 2002/0045643, International Publication No. WO98/54170, and U.S. Pat. No. 6,395,754, each of which is incorporatedherein by reference. Representative compounds are of formula III:

and pharmaceutically acceptable salts, hydrates, solvates, clathrates,enantiomers, diastereomers, racemates, and mixtures of stereoisomersthereof, wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R is H or CH₂OCOR′;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹, R²,R³, or R⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbons

R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R′ is R⁷—CHR¹⁰—N(R⁸R⁹);

R⁷ is m-phenylene or p-phenylene or —(C_(n)H_(2n))— in which n has avalue of 0 to 4; each of R⁸ and R⁹ taken independently of the other ishydrogen or alkyl of 1 to 8 carbon atoms, or R⁸ and R⁹ taken togetherare tetramethylene, pentamethylene, hexamethylene, or —CH₂CH₂X₁CH₂CH₂—in which X₁ is —O—, —S—, or —NH—;

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

* represents a chiral-carbon center.

Other representative compounds are of formula:

wherein:

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, and R⁴ is —NHR⁵ and the remaining of R¹, R², R³, andR⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbon atoms;

R⁶ is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R⁷ is m-phenylene or p-phenylene or —(C_(n)H_(2n))— in which n has avalue of 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂X¹CH₂CH₂— in which X¹ is —O—,—S—, or —NH—;

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl.

Other representative compounds are of formula:

in which

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

each of R¹, R², R³, and R⁴, independently of the others, is halo, alkylof 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one ofR¹, R², R³, and R⁴ is nitro or protected amino and the remaining of R¹,R², R³, and R⁴ are hydrogen; and

R⁶ is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro.

Other representative compounds are of formula:

in which:

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

(i) each of R¹, R², R³, and R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, and R⁴ is —NHR⁵ and the remaining of R¹, R², R³, andR⁴ are hydrogen;

R⁵ is hydrogen, alkyl of 1 to 8 carbon atoms, or CO—R⁷—CH(R¹⁰)NR⁸R⁹ inwhich each of R⁷, R⁸, R⁹, and R¹⁰ is as herein defined; and

R⁶ is alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro.

Specific examples of the compounds are of formula:

in which:

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

R⁶ is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl, chloro, or fluoro;

R⁷ is m-phenylene, p-phenylene or —(C_(n)H_(2n))— in which n has a valueof 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂X¹CH₂CH₂— in which X¹ is —O—,—S— or —NH—; and

R₁₀ is hydrogen, alkyl of 1 to 8 carbon atoms, or phenyl.

Preferred immunomodulatory compounds of the invention are4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione and3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. Thecompounds can be obtained via standard, synthetic methods (see e.g.,U.S. Pat. No. 5,635,517, incorporated herein by reference). Thecompounds are available from Celgene Corporation, Warren, N.J.4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione has thefollowing chemical structure:

The compound3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione has thefollowing chemical structure:

In another embodiment, specific immunomodulatory compounds of theinvention encompass polymorphic forms of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione such as Form A, B, C, D, E,F, G and H, disclosed in U.S. provisional application No. 60/499,723filed on Sep. 4, 2003, and the corresponding U.S. non-provisionalapplication, filed Sep. 3, 2004, both of which are incorporated hereinby reference. For example, Form A of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is an unsolvated,crystalline material that can be obtained from non-aqueous solventsystems. Form A has an X-ray powder diffraction pattern comprisingsignificant peaks at approximately 8, 14.5, 16, 17.5, 20.5, 24 and 26degrees 2θ, and has a differential scanning calorimetry meltingtemperature maximum of about 270° C. Form A is weakly or not hygroscopicand appears to be the most thermodynamically stable anhydrous polymorphof 3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dionediscovered thus far.

Form B of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is a hemihydrated,crystalline material that can be obtained from various solvent systems,including, but not limited to, hexane, toluene, and water. Form B has anX-ray powder diffraction pattern comprising significant peaks atapproximately 16, 18, 22 and 27 degrees 2θ, and has endotherms from DSCcurve of about 146 and 268° C., which are identified dehydration andmelting by hot stage microscopy experiments. Interconversion studiesshow that Form B converts to Form E in aqueous solvent systems, andconverts to other forms in acetone and other anhydrous systems.

Form C of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is a hemisolvatedcrystalline material that can be obtained from solvents such as, but notlimited to, acetone. Form C has an X-ray powder diffraction patterncomprising significant peaks at approximately 15.5 and 25 degrees 2θ,and has a differential scanning calorimetry melting temperature maximumof about 269° C. Form C is not hygroscopic below about 85% RH, but canconvert to Form B at higher relative humidities.

Form D of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is a crystalline, solvatedpolymorph prepared from a mixture of acetonitrile and water. Form D hasan X-ray powder diffraction pattern comprising significant peaks atapproximately 27 and 28 degrees 2θ, and has a differential scanningcalorimetry melting temperature maximum of about 270° C. Form D iseither weakly or not hygroscopic, but will typically convert to Form Bwhen stressed at higher relative humidities.

Form E of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is a dihydrated, crystallinematerial that can be obtained by slurrying 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione in water and by a slowevaporation of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione in a solvent system with aratio of about 9:1 acetone:water. Form E has an X-ray powder diffractionpattern comprising significant peaks at approximately 2θ, 24.5 and 29degrees 2θ, and has a differential scanning calorimetry meltingtemperature maximum of about 269° C. Form E can convert to Form C in anacetone solvent system and to Form G in a THF solvent system. In aqueoussolvent systems, Form E appears to be the most stable form. Desolvationexperiments performed on Form E show that upon heating at about 125° C.for about five minutes, Form E can convert to Form B. Upon heating at175° C. for about five minutes, Form B can convert to Form F.

Form F of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is an unsolvated,crystalline material that can be obtained from the dehydration of FormE. Form F has an X-ray powder diffraction pattern comprising significantpeaks at approximately 19, 19.5 and 25 degrees 2θ, and has adifferential scanning calorimetry melting temperature maximum of about269° C.

Form G of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is an unsolvated,crystalline material that can be obtained from slurrying forms B and Ein a solvent such as, but not limited to, tetrahydrofuran (THF). Form Ghas an X-ray powder diffraction pattern comprising significant peaks atapproximately 21, 23 and 24.5 degrees 2θ, and has a differentialscanning calorimetry melting temperature maximum of about 267° C.

Form H of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is a partially hydrated(about 0.25 moles) crystalline material that can be obtained by exposingForm E to 0% relative humidity. Form H has an X-ray powder diffractionpattern comprising significant peaks at approximately 15, 26 and 31degrees 2θ, and has a differential scanning calorimetry meltingtemperature maximum of about 269° C.

Other specific immunomodulatory compounds of the invention include, butare not limited to,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3yl)isoindolines and1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl)isoindolines such asthose described in U.S. Pat. Nos. 5,874,448 and 5,955,476, each of whichis incorporated herein by reference. Representative compounds are offormula:

wherein Y is oxygen or H² and

each of R¹, R², R³, and R⁴, independently of the others, is hydrogen,halo, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, oramino.

Other specific immunomodulatory compounds of the invention include, butare not limited to, the tetra substituted2-(2,6-dioxopiperdin-3-yl)-1-oxoisoindolines described in U.S. Pat. No.5,798,368, which is incorporated herein by reference. Representativecompounds are of formula:

wherein each of R¹, R², R³, and R⁴, independently of the others, ishalo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms.

Other specific immunomodulatory compounds of the invention include, butare not limited to, 1-oxo and1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindolines disclosed in U.S. Pat.No. 6,403,613, which is incorporated herein by reference. Representativecompounds are of formula:

in which:

Y is oxygen or H₂,

a first of R¹ and R² is halo, alkyl, alkoxy, alkylamino, dialkylamino,cyano, or carbamoyl, the second of R¹ and R², independently of thefirst, is hydrogen, halo, alkyl, alkoxy, alkylamino, dialkylamino,cyano, or carbamoyl, and

R³ is hydrogen, alkyl, or benzyl.

Specific examples of the compounds are of formula:

wherein a first of R¹ and R² is halo, alkyl of from 1 to 4 carbon atoms,alkoxy of from 1 to 4 carbon atoms, dialkylamino in which each alkyl isof from 1 to 4 carbon atoms, cyano, or carbamoyl,

the second of R¹ and R², independently of the first, is hydrogen, halo,alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms,alkylamino in which alkyl is of from 1 to 4 carbon atoms, dialkylaminoin which each alkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl,and

R³ is hydrogen, alkyl of from 1 to 4 carbon atoms, or benzyl.

Specific examples include, but are not limited to,1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline.

Other representative compounds are of formula:

wherein a first of R¹ and R² is halo, alkyl of from 1 to 4 carbon atoms,alkoxy of from 1 to 4 carbon atoms, dialkylamino in which each alkyl isof from 1 to 4 carbon atoms, cyano, or carbamoyl,

the second of R¹ and R², independently of the first, is hydrogen, halo,alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms,alkylamino in which alkyl is of from 1 to 4 carbon atoms, dialkylaminoin which each alkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl,and

R³ is hydrogen, alkyl of from 1 to 4 carbon atoms, or benzyl.

Specific examples include, but are not limited to,1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline.

Other specific immunomodulatory compounds include, but are not limitedto, 1-oxo and 1,3-dioxoisoindolines substituted in the 4- or 5-positionof the indoline ring described in U.S. Pat. No. 6,380,239 and co-pendingU.S. application Ser. No. 10/900,270, filed Jul. 28, 2004, which areincorporated herein by reference. Representative compounds are offormula:

in which the carbon atom designated C* constitutes a center of chirality(when n is not zero and R¹ is not the same as R²); one of X¹ and X² isamino, nitro, alkyl of one to six carbons, or NH-Z, and the other of X¹or X² is hydrogen; each of R¹ and R² independent of the other, ishydroxy or NH-Z; R³ is hydrogen, alkyl of one to six carbons, halo, orhaloalkyl; Z is hydrogen, aryl, alkyl of one to six carbons, formyl, oracyl of one to six carbons; and n has a value of 0, 1, or 2; providedthat if X¹ is amino, and n is 1 or 2, then R¹ and R² are not bothhydroxy; and the salts thereof.

Further representative compounds are of formula:

in which the carbon atom designated C* constitutes a center of chiralitywhen n is not zero and R¹ is not R²; one of X¹ and X² is amino, nitro,alkyl of one to six carbons, or NH-Z, and the other of X¹ or X² ishydrogen; each of R¹ and R² independent of the other, is hydroxy orNH-Z; R³ is alkyl of one to six carbons, halo, or hydrogen; Z ishydrogen, aryl or an alkyl or acyl of one to six carbons; and n has avalue of 0, 1, or 2.

Specific examples include, but are not limited to,2-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-carbamoyl-butyric acid and4-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-cabamoyl-butyric acid,which have the following structures, respectively, and pharmaceuticallyacceptable salts, solvates, prodrugs, and stereoisomers thereof:

Other representative compounds are of formula:

in which the carbon atom designated C* constitutes a center of chiralitywhen n is not zero and R¹ is not R²; one of X¹ and X² is amino, nitro,alkyl of one to six carbons, or NH-Z, and the other of X¹ or X² ishydrogen; each of R¹ and R² independent of the other, is hydroxy orNH-Z; R³ is alkyl of one to six carbons, halo, or hydrogen; Z ishydrogen, aryl, or an alkyl or acyl of one to six carbons; and n has avalue of 0, 1, or 2; and the salts thereof.

Specific examples include, but are not limited to,4-carbamoyl-4-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-butyricacid,4-carbamoyl-2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-butyricacid,2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-4-phenylcarbamoyl-butyricacid, and2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-pentanedioicacid, which have the following structures, respectively, andpharmaceutically acceptable salts, solvate, prodrugs, and stereoisomersthereof:

Other specific examples of the compounds are of formula:

wherein one of X¹ and X² is nitro, or NH-Z, and the other of X¹ or X² ishydrogen;

each of R¹ and R², independent of the other, is hydroxy or NH-Z;

R³ is alkyl of one to six carbons, halo, or hydrogen;

Z is hydrogen, phenyl, an acyl of one to six carbons, or an alkyl of oneto six carbons; and

n has a value of 0, 1, or 2;

provided that if one of X¹ and X² is nitro, and n is 1 or 2, then R¹ andR² are other than hydroxy; and

if —COR² and —(CH₂)_(n)COR¹ are different, the carbon atom designated C*constitutes a center of chirality.

Other representative compounds are of formula:

wherein one of X¹ and X² is alkyl of one to six carbons;

each of R¹ and R², independent of the other, is hydroxy or NH-Z;

R³ is alkyl of one to six carbons, halo, or hydrogen;

Z is hydrogen, phenyl, an acyl of one to six carbons, or an alkyl of oneto six carbons; and

n has a value of 0, 1, or 2; and

if —COR² and —(CH₂)_(n)COR¹ are different, the carbon atom designated C*constitutes a center of chirality.

Still other specific immunomodulatory compounds of the inventioninclude, but are not limited to, isoindoline-1-one andisoindoline-1,3-dione substituted in the 2-position with2,6-dioxo-3-hydroxypiperidin-5-yl described in U.S. Pat. No. 6,458,810,which is incorporated herein by reference. Representative compounds areof formula:

wherein:

the carbon atoms designated * constitute centers of chirality;

X is —C(O)— or —CH₂—;

R¹ is alkyl of 1 to 8 carbon atoms or —NHR³;

R² is hydrogen, alkyl of 1 to 8 carbon atoms, or halogen;

and

R³ is hydrogen,

alkyl of 1 to 8 carbon atoms, unsubstituted or substituted with alkoxyof 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbonatoms,

cycloalkyl of 3 to 18 carbon atoms,

phenyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms,alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4carbon atoms,

benzyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms,alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4carbon atoms, or —COR⁴ in which

R⁴ is hydrogen,

alkyl of 1 to 8 carbon atoms, unsubstituted or substituted with alkoxyof 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbonatoms,

cycloalkyl of 3 to 18 carbon atoms,

phenyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms,alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4carbon atoms, or

benzyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms,alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4carbon atoms.

Compounds of the invention can either be commercially purchased orprepared according to the methods described in the patents or patentpublications disclosed herein. Further, optically pure compounds can beasymmetrically synthesized or resolved using known resolving agents orchiral columns as well as other standard synthetic organic chemistrytechniques.

The composition of the invention may also comprise an immunomodulatorycompound as described in United States Application Publication No.2003/0235909, which is incorporated here by reference in its entirety.

5.1.4 Vasodilators

In another embodiment, the stem cell collection composition of theinvention comprises a vasodilator. The stem cell collection compositioncomprising a vasodilator is particularly useful for the collectionplacenta-derived stem cells by perfusion of the mammalian placenta. Thevasodilator may be any vasodilator known in the art, includingnaturally-occurring vasodilators and artificial vasodilators. In oneembodiment, the vasodilator is an antihypertensive drug. Such anantihypertensive drug can be a drug that activates guanylyl cyclase,ADP-ribosyl transferase or cyclooxygenase, or all three, and/or inhibitslipoxygenase. The vasodilator can be an organic or inorganic compound,e.g., atrial natriuretic peptide (ANP), adrenocorticotropin,corticotropin-releasing hormone, sodium nitroprusside, hydralazine,adenosine triphosphate, adenosine, indomethacin or magnesium sulfate,and the like. In other embodiment, the vasodilator is aphosphodiesterase inhibitor (e.g., dibutyryl adenosine,isobutylmethylxanthine, indolidan, roliprarn,2-o-propoxyphenyl-8-azapurin-6-one, trequensin, amrinone, milrinon,aminophylline or dipyridamole).

For stem cell collection by perfusion, hydralazine can be used in aconcentration of from about 0.1 mM to about 10 mM. Likewise, adenosinecan be used at a concentration of about 0.001 mM to about 10.0 mM;adenosine triphosphate can be used at a concentration of about 0.1 mM toabout 1000 mM; indomethacin can be used at a concentration of about 1mg/kg to about 20 mg/kg, wherein “kg” is the weight of an organ, e.g.,placenta; or magnesium sulfate can be used at a concentration of about0.1 mM to about 20 mM.

5.1.5 Inhibitors of Necrosis

The invention further provides a stem cell collection composition thatcomprises an inhibitor of necrosis. The inhibitor of necrosis can be anyphysiologically-acceptable necrosis inhibitor known in the art.

Thus, in one embodiment, the invention provides a stem cell collectioncomposition comprising, in a physiologically-acceptable solution, aninhibitor of necrosis. In another embodiment, the invention provides astem cell collection composition comprising, in aphysiologically-acceptable solution, an inhibitor of necrosis and aninhibitor of apoptosis. In a specific embodiment, the inhibitor ofnecrosis is 2-(1H-Indol-3-yl)-3-pentylamino-maleimide, pyrrolidinedithiocarbamate, or clonazepam. In other specific embodiments, the stemcell collection composition comprising a necrosis inhibitor, or aninhibitor of necrosis and an inhibitor of apoptosis, can comprise one ormore enzymes, e.g., one or more of the enzymes described in Section5.1.1, above; an inhibitor of apoptosis described in Section 5.1.2,above; an immunomodulatory compound, e.g., an immunomodulatory compounddescribed in Section 5.1.3, above; a vasodilator, e.g., a vasodilatordescribed in Section 5.1.4, above, or an oxygen-carryingperfluorocarbon, e.g., an oxygen-carrying perfluorocarbon described inSection 5.1.6, below.

In one embodiment, the invention provides a method of isolating a stemcell, comprising contacting said stem cell with a solution comprising aninhibitor of necrosis, and isolating said stem cell. In anotherembodiment, the invention provides a method of isolating a stem cellcomprising contacting said stem cell with a solution comprising aninhibitor of necrosis and an inhibitor of apoptosis, and isolating saidstem cell. In specific embodiments, the inhibitor of necrosis is2-(1H-Indol-3-yl)-3-pentylamino-maleimide, pyrrolidine dithiocarbamate,clonazepam, and the like.

5.1.6 Oxygen-Carrying Perfluorocarbons

The stem cell collection composition of the invention can furthercomprise one or more oxygen-carrying perfluorocarbons. Generally,oxygen-carrying perfluorocarbons are not water miscible, and the stemcell collection composition is otherwise generally aphysiologically-acceptable aqueous solution. The stem cell collectioncomposition comprising an oxygen-carrying perfluorocarbon therefore cancomprise separate aqueous and perfluorocarbon phases, or can compriseboth phases as an emulsion. Stem cells may be contacted duringcollection with either a two-phase or an emulsified composition, or maybe collected with the aqueous phase followed by contact with theperfluorocarbon phase. For example, stem cells in an organ, e.g., aplacenta, can be contacted during perfusion or tissue disruption, e.g.,enzymatic digestion, with a physiologically-acceptable aqueous solutioncomprising an inhibitor of apoptosis, e.g., a JNK inhibitor, and theresulting cell suspension combined with a perfluorocarbon. Thephysiologically acceptable solution comprising an apoptosis inhibitorand the perfluorocarbon may also be mixed, e.g., emulsified, prior tocollection of stem cells. The emulsion can then be used, e.g., as aperfusion solution, or a solution into which cells from a disruptedorgan, or portion thereof, can be suspended.

Thus, in one embodiment, said apoptosis inhibitor and saidperfluorocarbon are contained within a single solution, e.g., anemulsion, prior to contacting stem cells to be preserved. In anotherembodiment, said apoptosis inhibitor is contained within a firstsolution, and said perfluorocarbon is contained within a secondsolution, prior to said contacting.

The oxygen-carrying perfluorocarbon can comprise a single species ofperfluorocarbon, or can comprise a plurality of species. For example,the oxygen-carrying perfluorocarbon can comprise a perfluoroalkyl orperfluoroether. In specific embodiments, the oxygen-carryingperfluorocarbon can comprise one or more of an aliphatic perfluorocarbonof the general structure C_(n)F_(2n+1)R or C_(n)F_(2n)R₂, where n is aninteger from 8 to 12 and R is a lipophilic moiety; a perfluoroether ofthe general structure C_(n)F_(2n)+1−O−C_(n).F_(2n+1);perflourooctylethane, perfluorooctyldecane, perfluorodecalin,perfluoromethylbicyclo [3.3.1]-nonane, perfluorodimethyl bicyclononane,perfluoro-2,2,4,4-tetramethylpentane, perfluorotripropylamine,bis(F-butyl)ethene, (F-isopropyl) (F-hexyl)ethene,perfluoromethyladamantane, perfluorodimethyladamantane,F—N-methyldecahydroisoquinoline, F-4-methyloctahydroquinolidizine,perfluorooctyl bromide, perfluorodecyl bromide, α,ω-dichloro-F-decane,α,ω-dibromo-F-decane, C₁₀F₂₁CH═CH₂, C₁₀F₂,CH₂CH₃, and the like.

The oxygen-carrying perfluorocarbon may be present in the stem cellcollection composition may be present in an emulsion in an amountrepresenting about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, orabout 125% weight/volume of the emulsion. Preferably, the stem cellcollection composition comprising an oxygen-carrying perfluorocarbonadditionally comprises an emulsifier (e.g., a surfactant). Such anemulsifier can be any physiologically-acceptable emulsifier, and ispreferably lecithin. The emulsifier can be present in an amount of about0.1% to about 7% (w/v).

In a preferred embodiment, the stem cell collection compositioncomprises a first perfluorocarbon and a second perfluorocarbon having ahigher molecular weight than the first perfluorocarbon. Typically, thetotal volume of perfluorocarbon comprises about 50% to about 99.9% ofthe first perfluorocarbon, and about 0.1% to about 50% of the secondperfluorocarbon. In one embodiment, the first perfluorocarbon isperfluorooctyl bromide, and the second perfluorocarbon is perfluorodecylbromide.

The oxygen-carrying perfluorocarbon and aqueous portion of the stem cellcollection composition can be emulsified using standard emulsificationtechniques, e.g., high-pressure homogenization as described in U.S. Pat.No. 4,987,154; shaking in a flask; mechanical or ultrasonicemulsification of an emulsion formulation in a Manton-Gaulin mixer orMicrofluidizer (Microfluidics Corp., Newton, Mass.); etc. Where the stemcell collection composition comprises two or more distinct species ofoxygen-carrying perfluorocarbon, the species can be combined with theaqueous phase in the desired ratio, together with the emulsifier.Usually, a preemulsion mixture is prepared by simple mixing or blendingof the various components. This preemulsion is then emulsified in thedesired emulsification apparatus. The emulsification technique, wherethe stem cells are collected in a two-phase solution, is preferablyselected so as to minimize damage to the stem cells.

Apart from inclusion within a stem cell collection composition,oxygen-carrying perfluorocarbons can also be used in the preservation ofa placenta during transport from the site of placental collection (e.g.,delivery room) to the site of stem cell collection. In one embodiment, aplacenta, from which stem cells are to be collected, is contacted with acomposition comprising one or more oxygen-carrying perfluorocarbons forat least part of the time between placental collection and stem cellcollection. Preferably, the placenta is kept in contact with theoxygen-carrying perfluorocarbon for a majority of the time betweenplacental collection and stem cell collection. In another embodiment,the placenta is contacted with a composition comprising one or moreoxygen-carrying perfluorocarbons and one or more organ preservationmedia (e.g., UW solution; see Section 5.3, below) for at least part ofthe time between placental collection and stem cell collection.

5.1.7 Other Components

The stem cell collection composition can comprise other components thatcan reduce injury to the cell caused by an aspect of collection orstorage, organismal contamination, etc., or that can facilitate thecollection of the stem cells.

The stem cell collection composition can comprise a stem cell mobilizerand/or stimulator of hematopoiesis, such as a VLA-4 (Very Late Antigen)antagonist (e.g., an alpha-4 integrin antagonist, such as an antibody,e.g., Natalizumab or Anti-phospho-Integrin α4 (Ser988), clone 6.33(Upstate Cell Signaling Solutions), or a peptide (e.g.,phenylacetyl-leu-asp-phe-D-prolineamide (Cytel Corp., San DiegoCalif.))) at, e.g., 1-10 mg/kg of placenta weight, a CXCR-4 agonist(e.g., MOZOBIL™ (also known as AMD3100; AnorMED, Langley, BC, Canada)at, e.g., 0.01-10 mg/kg, alone or with G-CSF; SDF-1 (stromalcell-derived factor) analogs (e.g., CTCE-0214 from ChemokineTherapeutics Corp.) or an anti-SDF-1 antibody at, e.g., 0.01-10 mg/kg,and the like.

The stem cell collection composition can comprise a bacteriocidally orbacteriostatically effective amount of an antibiotic. In certainnon-limiting embodiments, the antibiotic is a macrolide (e.g.,tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime,cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, anerythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g.,ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, etc. In aparticular embodiments, the antibiotic is active against Gram(+) and/orGram(−) bacteria, e.g., Pseudomonas aeruginosa, Staphylococcus aureus,and the like.

The stem cell collection composition can comprise one or more of thefollowing compounds: adenosine (about 1 mM to about 50 mM); D-glucose(about 20 mM to about 100 mM); magnesium ions (about 1 mM to about 50mM); a macromolecule of molecular weight greater than 20,000 daltons, inone embodiment, present in an amount sufficient to maintain endothelialintegrity and cellular viability (e.g., a synthetic or naturallyoccurring colloid, a polysaccharide such as dextran or a polyethyleneglycol present at about 25 g/l to about 100 g/l, or about 40 g/l toabout 60 g/l); an antioxidant (e.g., butylated hydroxyanisole, butylatedhydroxytoluene, glutathione, vitamin C or vitamin E present at about 25μM to about 100 μM); a reducing agent (e.g., N-acetylcysteine present atabout 0.1 mM to about 5 mM); an agent that prevents calcium entry intocells (e.g., verapamil present at about 2 μM to about 25 μM);nitroglycerin (e.g., about 0.05 g/L to about 0.2 g/L); an anticoagulant,in one embodiment, present in an amount sufficient to help preventclotting of residual blood (e.g., heparin or hirudin present at aconcentration of about 1000 units/l to about 100,000 units/l); or anamiloride containing compound (e.g., amiloride, ethyl isopropylamiloride, hexamethylene amiloride, dimethyl amiloride or isobutylamiloride present at about 1.0 μM to about 5 μM).

5.2 Methods of Collecting Stem Cells Using the Compositions of theInvention

The invention further provides methods of collecting and isolatingplacental stem cells using the stem cell collection compositiondescribed above.

In one embodiment, the invention provides a method of isolating a stemcell, comprising contacting said stem cell with a solution comprising aninhibitor of necrosis, e.g., an inhibitor of necrosis described inSection 5.1.5, above, and isolating said stem cell. In anotherembodiment, the invention provides a method of isolating a stem cell,comprising contacting said stem cell with a solution comprising aninhibitor of apoptosis, e.g., an inhibitor of apoptosis described inSection 5.1.2, above, and isolating said stem cell. In anotherembodiment, the invention provides a method of isolating a stem cellcomprising contacting said stem cell with a solution comprising aninhibitor of necrosis e.g., an inhibitor of necrosis described inSection 5.1.5, above and an inhibitor of apoptosis, e.g., an inhibitorof apoptosis described in Section 5.1.2, above, and isolating said stemcell. In specific embodiments, the inhibitor of necrosis is2-(1H-Indol-3-yl)-3-pentylamino-maleimide, pyrrolidine dithiocarbamate,clonazepam, and the like. In a specific embodiment, the inhibitor ofapoptosis is a caspase inhibitor or a c-Jun N-terminal kinase (JNK)inhibitor. In another specific embodiment, the JNK inhibitor does notmodulate the differentiation or proliferation of said stem cell prior tosaid isolation; that is, the JNK inhibitor does not detectably alter thedifferentiation or proliferation of the stem cell during contact withthe JNK inhibitor. The stem cell in the method can be isolated frommammalian placenta, umbilical cord, placental blood or umbilical cordblood. The method also encompasses contacting the stem cell with anoxygen-carrying perfluorocarbon, e.g., an oxygen-carryingperfluorocarbon described in Section 5.1.6. In another embodiment, themethod comprises contacting said stem cell with an enzyme, e.g., aprotease. The protease can be, e.g., a matrix metalloprotease or aneutral protease, and can be, for example, collagenase, thermolysin ordispase. The stem cells can, in another embodiment, be contacted with amucolytic enzyme, such as hyaluronidase.

The stem cell collection composition used in the method can comprise asolution, for example, a saline solution or culture medium. In someembodiments, the solution comprises hydroxyethyl starch, lactobionicanion and raffinose, and/or UW solution.

In preferred embodiments, the stem cell is not exposed to hypoxic orshear stress during isolation, or such stresses are minimized to theextent possible. For example, in one embodiment, the stem cell isexposed during isolation to a hypoxic condition for less than six hours,less than two hours, less than one hour, or less than thirty minutesduring said isolation, wherein a hypoxic condition is a concentration ofoxygen that is less than normal blood oxygen concentration.

5.2.1 Physical Disruption

In one embodiment, stem cells are collected from a mammalian organ, e.g.placenta, by physical disruption, e.g., enzymatic digestion, of theorgan. For example, the organ, or a portion thereof, may be, e.g.,crushed, sheared, minced, diced, chopped, macerated or the like, whilein contact with the stem cell collection composition of the invention,and the tissue subsequently digested with one or more enzymes. Theorgan, or a portion thereof, may also be physically disrupted anddigested with one or more enzymes, and the resulting material thenimmersed in, or mixed into, the stem cell collection composition of theinvention. The stem cell collection composition of the invention cancomprise the enzyme(s). Any method of physical disruption can be used,provided that the method of disruption leaves a plurality, preferably amajority, preferably at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%of the cells in said organ viable, as determined by, e.g., trypan blueexclusion. Preferably, the method of disruption leaves a plurality,preferably a majority, preferably at least 50%, 60%, 70%, 80%, 90%, 95%,98%, or 99% of the stem cells in said organ viable, as determined by,e.g., trypan blue exclusion.

Any combination of enzymes can be used, e.g., one or more of theproteases and/or mucolytic enzymes disclosed in Section 5.1.1, above.For example, placental tissue may be digested using collagenase (e.g.,collagenase I, II, III or IV), dispase, elastase, trypsin etc. alone, orany combination thereof. Typical concentrations for tissue digestionenzymes include, e.g., 50-200 u/mL for collagenase I and collagenase IV,1-10 u/mL for dispase, and 10-100 u/mL for elastase. Proteases can beused in combination, that is, two or more proteases in the samedigestion reaction, or can be used sequentially in order to liberateplacental stem cells. For example, in one embodiment, a placenta, orpart thereof, is digested first with an appropriate amount ofcollagenase I at 2 mg/ml for 30 minutes, followed by digestion withtrypsin, 0.25%, for 10 minutes, at 37° C.

In another embodiment, the tissue can further be disrupted by theaddition of a chelator, e.g., ethylene glycol bis(2-aminoethylether)-N,N,N′N′-tetraacetic acid (EGTA) or ethylenediaminetetraaceticacid (EDTA) to the stem cell collection composition comprising the stemcells, or to a solution in which the tissue is disrupted and/or digestedprior to isolation of the stem cells with the stem cell collectioncomposition.

5.2.2 Perfusion

Cells, e.g., stem cells, can be collected from an organ, e.g., amammalian placenta, by perfusion, e.g., through the placentalvasculature, using the stem cell collection composition of the inventionas a perfusion solution. In one embodiment, a mammalian placenta isperfused by passage of perfusion solution through either or both of theumbilical artery and umbilical vein (FIG. 1, FIGS. 2A-2D). The flow ofperfusion solution through the placenta may be accomplished using, e.g.,gravity flow into the placenta. Preferably, the perfusion solution isforced through the placenta using a pump, e.g., a peristaltic pump. Theumbilical vein can be, e.g., cannulated with a cannula, e.g., a TEFLON®or plastic cannula, that is connected to a sterile connection apparatus,such as sterile tubing. The sterile connection apparatus is connected toa perfusion manifold, as shown in FIG. 3.

Perfusion can also be aided by the use of a pressure chamber, e.g., achamber in which the pressure of the atmosphere surrounding the placentaduring perfusion is greater than atmospheric pressure. In oneembodiment, the pressure chamber, or tank, comprises a plurality ofports that allow ingress and egress of a plurality of tubes. Such tubescan be connected to placental veins and arteries so as to facilitateperfusion by collection of exudate of perfusate from the placenta, or ofperfusate that has been circulated only through the placentalvasculature. The tank also comprises a port, connected to a pump thatcontrols the atmospheric pressure inside the tank. In a specificexample, perfusion fluid is pumped into the placental (fetal)vasculature using one pump, and a second pump is used to controlatmospheric pressure against the placenta to, for example, applypressure to aid in collecting perfusate, or to mimic the naturalpressure against the placenta as when the placenta is carried in vivo.

In preparation for perfusion, the placenta is preferably oriented (e.g.,suspended) in such a manner that the umbilical artery and umbilical veinare located at the highest point of the placenta. The placenta can beperfused by passage of a perfusion fluid, e.g., the stem cell collectioncomposition of the invention, through the placental vasculature, orthrough the placental vasculature and surrounding tissue. Perfusionsolution can be passed through any of the umbilical vessels, in anycombination. Perfusion solution can be passed through the placentalvasculature in either direction (that is, perfusion solution can beperfused into a placental artery or into a placental vein, or collectedfrom a placental artery or a placental vein, or from seepage fromplacental tissue). In one embodiment, the umbilical artery and theumbilical vein are connected simultaneously, as shown in FIG. 1, to apipette that is connected via a flexible connector to a reservoir of theperfusion solution. The perfusion solution is passed into the umbilicalvein and artery. The perfusion solution exudes from and/or passesthrough the walls of the blood vessels into the surrounding tissues ofthe placenta, and is collected in a suitable open vessel from thesurface of the placenta that was attached to the uterus of the motherduring gestation. The perfusion solution may also be introduced throughthe umbilical cord opening and allowed to flow or percolate out ofopenings in the wall of the placenta which interfaced with the maternaluterine wall. In another embodiment, the perfusion solution is passedthrough the umbilical veins and collected from the umbilical artery, oris passed through the umbilical artery and collected from the umbilicalveins.

In one embodiment, the proximal umbilical cord is clamped duringperfusion, and more preferably, is clamped within 4-5 cm (centimeter) ofthe cord's insertion into the placental disc.

The first collection of perfusion fluid, e.g., stem cell collectioncomposition of the invention, from a mammalian placenta during theexsanguination process is generally colored with residual red bloodcells of the cord blood and/or placental blood. The perfusion fluidbecomes more colorless as perfusion proceeds and the residual cord bloodcells are washed out of the placenta. Generally from 30 to 100 ml(milliliter) of perfusion fluid is adequate to initially exsanguinatethe placenta, but more or less perfusion fluid may be used depending onthe observed results.

The volume of perfusion liquid used to collect placental stem cells mayvary depending upon the number of stem cells to be collected, the sizeof the placenta, the number of collections to be made from a singleplacenta, etc. In various embodiments, the volume of perfusion liquidmay be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mLto 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL.Typically, the placenta is perfused with 700-800 mL of perfusion liquidfollowing exsanguination. Because the invention contemplated theperfusion of any mammalian placenta, the placenta can be perfused withat least, or no more than, 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, 5.0 mL, 6.0mL, 7.0 mL, 8.0 mL, 9.0 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40mL, 45 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 85 mL 90 mL,95 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL 600 mL, 700 mL, 800 mL,900 mL, 1000 mL, 1500 mL, 2000 mL, 2500 mL, 3000 mL, 3500 mL, 4000 mL,4500 mL, 5000 mL, 5500 mL, 6000 mL, 6500 mL, 7000 mL, 7500 mL, 8000 mL,8500 mL, 9000 mL, 9500 mL, or 10,000 mL of perfusion liquid to collectplacenta-derived stem cells.

The organ, e.g. placenta, can be perfused a plurality of times over thecourse of several hours or several days. Where the organ is to beperfused a plurality of times, it may be maintained or cultured underaseptic conditions in a container or other suitable vessel, and perfusedwith the stem cell collection composition of the invention, or astandard perfusion solution (e.g., a normal saline solution such asphosphate buffered saline (“PBS”)) with or without an anticoagulant(e.g., heparin, warfarin sodium, coumarin, bishydroxycoumarin, citrate,citrate phosphate dextrose adenine (CPDA-1)), and/or with or without anantimicrobial agent (e.g., antibiotics such as streptomycin (e.g., at40-100 μg/ml), penicillin (e.g., at 40 U/ml), amphotericin B (e.g., at0.5 μg/ml). In one embodiment, an isolated organ, e.g. placenta, ismaintained or cultured for a period of time without collecting theperfusate, such that the organ is maintained or cultured for 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,or 24 hours, or 2 or 3 or more days before perfusion and collection ofperfusate. The perfused organ may be maintained for one or moreadditional time(s), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and perfused asecond time with, e.g., 700-800 mL perfusion fluid. The organ, e.g.,placenta, may be perfused 1, 2, 3, 4, 5 or more times, for example, onceevery 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment, perfusion ofthe placenta and collection of perfusion solution, e.g., stem cellcollection composition, is repeated until the number of recoverednucleated cells falls below 100 cells/ml. The perfusates at differenttime points can be further processed individually to recovertime-dependent populations of cells, e.g., stem cells. Perfusates fromdifferent time points can also be pooled.

The placenta can optionally be cultured in one or more of the stem cellcollection compositions described herein, wherein the stem cellcollection composition, rather than being immediately collected, isallowed to remain in the placental vasculature for a time prior tocollection of placenta-derived stem cells. For example, in oneembodiment, a placenta can be perfused with enough of a stem cellcollection composition to fill the placental vasculature, wherein thestem cell collection composition comprises, e.g., a vasodilator, whichcan facilitate subsequent collection of stem cells. In anotherembodiment, a placenta is perfused with a stem cell collectioncomposition comprising, e.g., one or more proteases. The placenta iscultured with the stem cell collection composition for a time sufficientfor the one or more proteases to begin digestion of the placenta tissue,e.g., 1-5 hours, whereupon the stem cells are collected by furtherperfusion.

The placenta can, as well, be perfused using different stem cellcollection compositions. For example, a placenta can first be perfusedwith a stem cell collection composition-comprising a vasodilator for atime sufficient for the vasodilator to dilate placental vessels.Preferably, only as much of the stem cell collection composition asneeded to fill the placental vasculature is used. The placenta canoptionally be cultured in the stem cell collection composition for atime prior to stem cell collection, as described above. The placenta isthen perfused with a second stem cell collection composition comprisingone or more proteases. The placenta can optionally be cultured for atime with this stem cell collection composition for a time sufficientfor the protease(s) to begin digestion of the placental tissue. Theplacenta can then be perfused with additional stem cell collectioncomposition, or with a third stem cell collection composition, e.g., astem cell collection composition comprising one or more apoptosis and/ornecrosis inhibitors. Other sequential combinations of different stemcell collection compositions will be easily appreciated by those ofskill in the art.

In one embodiment, for spontaneous vaginal delivery (SVD), the placentalvasculature is filled with the collection composition of the invention,and the placenta is bathed (e.g., immersed) in the solution, and stemcells are collected, e.g., by perfusion, at from about 20 to about 24hours after delivery. In another embodiment, the placenta is immersed inthe collection composition for at least part of the time betweendelivery and stem cell collection. In another embodiment, thevasculature of the placenta is filled with the collection compositionfor at least part of the time between delivery and stem cell collection.In another embodiment, the placenta is refrigerated (e.g., at about 0°C. to about 5° C. immediately after delivery up to the time ofcollection of stem cells, e.g., by perfusion, enzymatic digestion, orculture of placental tissue. In a specific embodiment, stem cells arecollected from the refrigerated placenta at from about 20 to about 24hours after delivery.

Without wishing to be bound by any theory, after exsanguination and asufficient time of perfusion of the placenta, placental stem cells arebelieved to migrate into the exsanguinated and perfused microcirculationof the placenta where, according to the methods of the invention, theyare collected, preferably by washing into a collecting vessel byperfusion. Perfusing the isolated placenta not only serves to removeresidual cord blood but also provide the placenta with the appropriatenutrients, including oxygen. The placenta may be cultivated and perfusedwith a similar solution which was used to remove the residual cord bloodcells, preferably, without the addition of anticoagulant agents.

Perfusion according to the methods of the invention results in thecollection of significantly more placental stem cells than the numberobtainable from a mammalian placenta not perfused with said solution,and not otherwise treated to obtain stem cells (e.g., by tissuedisruption, e.g., enzymatic digestion). In this context, “significantlymore” means at least 10% more. Perfusion according to the methods of theinvention yields significantly more placental stem cells than, e.g., thenumber of placental stem cells obtainable from culture medium in which aplacenta, or portion thereof, has been cultured.

Stem cells may be isolated from, e.g., placenta by perfusion with asolution comprising one or more proteases or other tissue-disruptiveenzymes. In a specific embodiment, a placenta or portion thereof (e.g.,amniotic membrane, amnion and chorion, placental lobule or cotyledon,umbilical cord, or combination of any of the foregoing) is brought to25-37° C., and is incubated with one or more tissue-disruptive enzymesin 200 mL of a culture medium for 30 minutes. Cells from the perfusateare collected, brought to 4° C., and washed with a cold inhibitor mixcomprising 5 mM EDTA, 2 mM dithiothreitol and 2 mM beta-mercaptoethanol.The stem cells are washed after several minutes with a cold (e.g., 4°C.) stem cell collection composition of the invention.

5.2.3 Isolation, Sorting and Characterization of Stem Cells

Cells from an organ, e.g., mammalian placenta, whether obtained byperfusion or enyzmatic digestion, can initially be purified from othercells (i.e., isolated) by Ficoll gradient centrifugation. Suchcentrifugation can follow any standard protocol for centrifugationspeed, etc. In one embodiment, cells collected from the organ, e.g.,placenta, are recovered from perfusate by centrifugation at about 400 toabout 1000×g for about 10-30 minutes at room temperature. In anotherembodiment, perfusate is concentrated to about 200 ml, gently layeredover, e.g., a Ficoll, Percoll or hetastarch gradient, and centrifuged atabout, e.g., 400-3000×g for 20 minutes at 22° C., and the low-densityinterface layer of cells is collected for further processing. In anotherembodiment, hetastarch is mixed with placental cells, and is allowed tosettle by gravity; the supernatant is removed and the hetastarch andcells are collected for further processing.

Cell pellets can be resuspended in fresh stem cell collectioncomposition, or a medium suitable for stem cell maintenance, e.g., IMDMserum-free medium containing 2 U/ml heparin and 2 mM EDTA (GibcoBRL,NY), DMEM, or the like. In one embodiment, placental cells obtained byperfusion or disruption, e.g., enzymatic digestion, can be selected inmedium composed of, e.g., DMEM-LG, MCDB-201, (ITS (insulin, transferrin,selenium), LA-BSA (linoleic acid-bovine serum albumin), dexamethasone,EGF (epidermal growth factor, PDGF (platelet-derived growth factor), andantibiotics, e.g., penicillin and/or streptomycin. The total mononuclearcell fraction can be isolated, e.g., using Lymphoprep (Nycomed Pharma,Oslo, Norway) according to the manufacturer's recommended procedure andthe mononuclear cell fraction was resuspended. The stem cellcomposition, or medium, in which the stem cells are resuspendedpreferably comprises a stem cell-compatible protein, e.g., albumin, forexample, human albumin.

As used herein, “isolating stem cells” means removing at least 50% ofthe cells with which the stem cells are normally associated with in theintact organ, e.g., placenta. A stem cell from an organ is “isolated”when it is present, with other stem cells from the organ, in apopulation of cells that comprises fewer than 50% of the cells withwhich the stem cell is normally associated in the intact organ.

Placental cells obtained by perfusion or digestion can be further, orinitially, isolated by differential trypsinization using, e.g., asolution of 0.05% trypsin with 0.2% EDTA (Sigma, St. Louis Mo.).Differential trypsinization is possible because fibroblastoid cellscollected from, e.g., placenta, typically detach from plastic surfaceswithin about five minutes whereas other adherent populations typicallyrequire more than 20-30 minutes incubation. The detached fibroblastoidcells can be harvested following trypsinization and trypsinneutralization, using, e.g., Trypsin Neutralizing Solution (TNS,Cambrex). In one embodiment of isolation of adherent cells, aliquots of,for example, about 5-10×10⁶ cells are placed in each of several T-75flasks, preferably fibronectin-coated T75 flasks. The cells are culturedwith commercially available Mesenchymal Stem Cell Growth Medium (MSCGM)Cambrex), and placed in a tissue culture incubator (37° C., 5% CO₂).After 10 to 15 days, non-adherent cells are removed from the flasks bywashing with PBS. The PBS is then replaced by MSCGM. Flasks arepreferably examined daily for the presence of various adherent celltypes and in particular, for identification and expansion of clusters offibroblastoid cells.

The number and type of cells collected from an organ, e.g., placenta,can easily be monitored by measuring changes in morphology and cellsurface markers using standard cell detection techniques such as flowcytometry, cell sorting, immunocytochemistry (e.g., staining with tissuespecific or cell-marker specific antibodies) fluorescence activated cellsorting (FACS), magnetic activated cell sorting (MACS), by examinationof the morphology of cells using light or confocal microscopy, or bymeasuring changes in gene expression using techniques well known in theart, such as PCR and gene expression profiling. Placental cells,particularly cells that have been isolated by Ficoll separation,differential adherence, or a combination of both, may be sorted using afluorescence activated cell sorter (FACS). Fluorescence activated cellsorting (FACS) is a well-known method for separating particles,including cells, based on the fluorescent properties of the particles(Kamarch, 1987, Methods Enzymol, 151:150-165). Laser excitation offluorescent moieties in the individual particles results in a smallelectrical charge allowing electromagnetic separation of positive andnegative particles from a mixture. In one embodiment, cell surfacemarker-specific antibodies or ligands are labeled with distinctfluorescent labels. Cells are processed through the cell sorter,allowing separation of cells based on their ability to bind to theantibodies used. FACS sorted particles may be directly deposited intoindividual wells of 96-well or 384-well plates to facilitate separationand cloning.

In one sorting scheme, stem cells from placenta are sorted on the basisof expression of the markers AC133, CD34, CD38, CD90, CD117, HLA-DR,CD10, CD4, CD71, CD38, CD45 and CD61 as shown in FIG. 4. In anotherembodiment, cells from placenta are based on their expression of markersCD200 and/or HLA-G; in one embodiment, cells displaying either of thesemarkers are isolated for further use. Cells that express, e.g., CD200and/or HLA-G can, in a specific embodiment, be further sorted based ontheir expression of CD73 or CD105, or epitopes recognized by antibodiesSH2, SH3 or SH4, or lack of expression of CD34, CD38 or CD45. Forexample, in one embodiment, placental cells are sorted by theirexpression, or lack thereof, of CD200, HLA-G, CD73, CD105, CD34, CD38and CD45, and placental cells that are CD200⁺, HLA-G⁺, CD73⁺, CD105⁺,CD34⁻, CD38⁻ and CD45⁻ are isolated from other placental cells forfurther use.

In another embodiment, magnetic beads can be used to separate cells. Thecells may be sorted using a magnetic activated cell sorting (MACS)technique, a method for separating particles based on their ability tobind magnetic beads (0.5-100 μm diameter). A variety of usefulmodifications can be performed on the magnetic microspheres, includingcovalent addition of antibody that specifically recognizes a particularcell surface molecule or hapten. The beads are then mixed with the cellsto allow binding. Cells are then passed through a magnetic field toseparate out cells having the specific cell surface marker. In oneembodiment, these cells can then isolated and re-mixed with magneticbeads coupled to an antibody against additional cell surface markers.The cells are again passed through a magnetic field, isolating cellsthat bound both the antibodies. Such cells can then be diluted intoseparate dishes, such as microtiter dishes for clonal isolation.

Placental cells, e.g., placental stem cells, can also be characterizedand/or sorted based on cell morphology and growth characteristics. Forexample, placental cells can be characterized as having, and/or selectedon the basis of, e.g., a cobblestone or fibroblastoid appearance inculture. Placental cells can also be characterized as having, and/orselected on the basis of, e.g., their ability or inability to formembryoid bodies. In one embodiment, for example, placental cells thatare fibroblastoid in shape, express CD73 and CD105, and produce one ormore embryoid bodies in culture are isolated from other placental cells.In another embodiment, OCT-4⁺ placental cells that produce one or moreembryoid bodies in culture are isolated from other placental cells.

In another embodiment, placental stem cells can be identified andcharacterized by a colony forming unit assay. Colony forming unitsassays are commonly known in the art, such as Mesen Cult™ medium (StemCell Technologies, Inc., Vancouver British Columbia)

Stem cells, e.g., placental stem cells, may be assessed for viability,proliferation potential, and longevity using standard techniques knownin the art, such as trypan blue exclusion assay, fluorescein diacetateuptake assay, propidium iodide uptake assay (to assess viability); andthymidine uptake assay, MTT cell proliferation assay (to assessproliferation). Longevity may be determined by methods well known in theart, such as by determining the maximum number of population doubling inan extended culture.

5.2.4 Stem Cell Storage

In other embodiments, the cells collected from the placenta arecryopreserved for use at a later time. Methods for cryopreservation ofcells, such as stem cells, are well known in the art, for example, Hu etal. (WO 00/73421, published Dec. 7, 2000).

5.2.5 Assessing Potency of Stem Cell Populations

A placenta-derived stem cell population, or population of isolated cellscomprising placenta-derived stem cells, as described herein can beassessed for potency, that is, the number of viable cells, using acolony-forming unit assay. In a preferred embodiment, the colony-formingunit assay is used to assess the potency of a placental stem cellpopulation contained within an initial perfusate of a placenta, or aninitial enzyme digestion of placental tissue. A colony-forming unitassay can also be performed on placenta-derived stem cell populations,or populations of isolated placental cells comprising placenta-derivedstem cells, that have been passaged at least once, at least five, ten,15, 20 or more times in culture. Any standard colony-forming unit assaycan be used, e.g., colony forming assays provided by StemCellTechnologies, Inc. Such an assay may use, e.g., MESENCULT™ medium (StemCell Technologies, Inc., Vancouver British Columbia).

The colony-forming unit assay can be performed on individual populations(e.g., individual units of perfusate or digestate) of placenta-derivedstem cells, or populations of isolated placental cells comprisingplacenta-derived stem cells, to assess the potency of the populations.For example, the colony-forming unit assay can be used, alone or withother tests, such as viability tests, to determine the number of usefulcells in a population of placenta-derived cells. Typically, thecolony-forming unit assay would be performed on a small sample cellsfrom of a population, and the results extrapolated to the remainingpopulation.

The colony-forming unit assay described above can be used to assess theefficacy of various formulations of the stem cell collectioncompositions, that is, can be used to identify stem cell collectionformulations that improve, or are optimal, for stem cell collectionunder varying circumstances. Such circumstances can include, forexample, methods of obtaining, transporting, culturing or handling aplacenta prior to stem cell collection; methods of collection of stemcells, time from placental collection to stem cell collection, presenceor absence of a placental culturing step as part of, or prior to, stemcell collection, and the like. In one embodiment, for example, theinvention provide a method of assessing the potency of aplacenta-derived stem cell population, or population of isolatedplacental cells comprising placenta-derived stem cells comprisingcontacting said population with a stem cell collection composition;determining a number of colony-forming units produced by said populationin a colony-forming unit assay; wherein said number of colony-formingunits is, or is correlated with, the potency of the population. In aspecific embodiment, the number of colony-forming units can be comparedto a control. For example, the control can be an absolute number ofcolony-forming units expected for a particular number ofplacenta-derived stem cells, e.g., a criterion for potency. In anotherexample, the control can be the number of colony-forming units producedby an equivalent number of placenta-derived stem cells contacted with asecond stem cell collection formulation; in this example, the methodallows for the comparison of two different formulations to assess whichis better at preserving, or producing higher numbers of viable,placenta-derived stem cells. In another specific embodiment, the controlis the number, or average number, of colony-forming units obtainableunder a particular method of transporting a placenta from site ofdelivery to site of stem cell collection. In another specificembodiment, said control is the number of colony-forming unitsobtainable under a particular method of handling the placenta at thesite of stem cell collection.

In another embodiment, the invention provides a method of selecting astem cell collection composition to preserve a population ofplacenta-derived stem cells, or population of isolated placental cellsthat comprises placenta-derived stem cells, comprising contacting afirst population comprising a number of placenta-derived stem cells witha first stem cell collection composition, wherein said first populationproduces a first number of colony-forming units in a colony-forming unitassay, and a second population comprising said number ofplacenta-derived stem cells with a second stem cell collectioncomposition, wherein said second population produces a second number ofcolony-forming units in a colony-forming unit assay, and comparing afirst number of colony-forming units to said second number ofcolony-forming units, and selecting whichever of said first number orsaid second number is higher.

5.3 Stem Cell Preservation

In another aspect, the invention provides compositions and methods forpreserving a population of stem cells. The invention encompasses thepreservation of a population of stem cells whether a the population is apopulation of isolated stem cells or a population of stem cells in situ,in an organ or tissue, prior to, or during, the process of isolation andcollection. For example, the methods and compositions of the inventioncan be used to preserve a population of isolated placental stem cells,or a population of placental stem cells in the placenta, e.g., while theplacenta is in transport or is awaiting processing to isolate andcollect placental stem cells.

The invention further provides methods of preserving a population ofstem cells using a stem cell collection composition comprising aninhibitor of apoptosis and an oxygen-carrying perfluorocarbon, e.g., aperfluorocarbon as described in Section 5.1.5, above. In one embodiment,the invention provides a method of preserving a population of stem cellscomprising contacting said population of stem cells with an inhibitor ofapoptosis and an oxygen-carrying perfluorocarbon, wherein said inhibitorof apoptosis is present in an amount and for a time sufficient to reduceor prevent apoptosis in the population of stem cells, as compared to apopulation of stem cells not contacted with the inhibitor of apoptosis.In a specific embodiment, said inhibitor of apoptosis is a caspaseinhibitor. In another specific embodiment, said inhibitor of apoptosisis a JNK inhibitor, e.g., a JNK inhibitor described in Section 5.1.2.2,above. In a more specific embodiment, said JNK inhibitor does notmodulate differentiation or proliferation of said stem cells. In anotherembodiment, said stem cell collection composition comprises saidinhibitor of apoptosis and said oxygen-carrying perfluorocarbon inseparate phases. In another embodiment, said stem cell collectioncomposition comprises said inhibitor of apoptosis and saidoxygen-carrying perfluorocarbon in an emulsion. In another embodiment,the stem cell collection composition additionally comprises anemulsifier, e.g., lecithin. In another embodiment, said apoptosisinhibitor and said perfluorocarbon are between about 0° C. and about 25°C. at the time of contacting the stem cells. In another more specificembodiment, said apoptosis inhibitor and said perfluorocarbon arebetween about 2° C. and 10° C., or between about 2° C. and about 5° C.,at the time of contacting the stem cells. In another more specificembodiment, said contacting is performed during transport of saidpopulation of stem cells. In another more specific embodiment, saidcontacting is performed during freezing and thawing of said populationof stem cells.

In another embodiment, the invention provides a method of preserving apopulation of stem cells comprising contacting said population of stemcells, or an organ (e.g., placenta) comprising the stem cells, with aninhibitor of apoptosis and an organ-preserving compound, wherein saidinhibitor of apoptosis is present in an amount and for a time sufficientto reduce or prevent apoptosis in the population of stem cells, ascompared to a population of stem cells not contacted with the inhibitorof apoptosis. In a specific embodiment, the organ-preserving compound isUW solution (descried in U.S. Pat. No. 4,798,824; also known as ViaSpan;see also Southard et al., Transplantation 49(2):251-257 (1990)) or asolution described in Stem et al., U.S. Pat. No. 5,552,267. In anotherembodiment, said organ-preserving compound is hydroxyethyl starch,lactobionic acid, raffinose, or a combination thereof. In anotherembodiment, the stem cell collection composition additionally comprisesan oxygen-carrying perfluorocarbon, either in two phases or as anemulsion.

In another embodiment of the method, said stem cells are contacted withsaid stem cell collection composition comprising an apoptosis inhibitorand oxygen-carrying perfluorocarbon, organ-preserving compound, orcombination thereof, during perfusion. In another embodiment, said stemcells are contacted during a process of tissue dissociation. In anotherembodiment, said stem cells are contacted with said stem cell collectioncompound after collection by perfusion, or after collection by tissuedisruption, e.g., enzymatic digestion.

Typically, during placental cell collection, enrichment and isolation,it is preferable to minimize or eliminate cell stress due to hypoxia andmechanical stress. In another embodiment of the method, therefore, astem cell, or population of stem cells, is exposed to a hypoxiccondition during collection, enrichment or isolation for less than 72hours during said preservation, wherein a hypoxic condition is aconcentration of oxygen that is less than normal blood oxygenconcentration. In a more specific embodiment, said population of stemcells is exposed to said hypoxic condition for less than 48 hours duringsaid preservation. In another more specific embodiment, said populationof stem cells is exposed to said hypoxic condition for less than 24hours, or less than 12, 6, or 2 hours, or is not exposed to a hypoxiccondition, during collection, enrichment or isolation. In anotherspecific embodiment, said population of stem cells is not exposed toshear stress during collection, enrichment or isolation.

5.4 Placental Handling

Generally, a human placenta is recovered shortly after its expulsionafter birth. In a preferred embodiment, the placenta is recovered from apatient after informed consent and after a complete medical history ofthe patient is taken and is associated with the placenta. Preferably,the medical history continues after delivery. Such a medical history canbe used to coordinate subsequent use of the placenta or the stem cellsharvested therefrom. For example, human placental stem cells can beused, in light of the medical history, for personalized medicine for theinfant associated with the placenta, or for parents, siblings or otherrelatives of the infant.

Prior to recovery of placental stem cells, the umbilical cord blood andplacental blood are removed. In certain embodiments, after delivery, thecord blood in the placenta is recovered. The placenta can be subjectedto a conventional cord blood recovery process. Typically a needle orcannula is used, with the aid of gravity, to exsanguinate the placenta(Anderson, U.S. Pat. No. 5,372,581; Hessel et al., U.S. Pat. No.5,415,665). The needle or cannula is usually placed in the umbilicalvein and the placenta can be gently massaged to aid in draining cordblood from the placenta. Such cord blood recovery may be obtainedcommercially, e.g., LifeBank Inc., Cedar Knolls, N.J., ViaCord, CordBlood Registry and Cryocell. Preferably, the placenta is gravity drainedwithout further manipulation so as to minimize tissue disruption duringcord blood recovery. In certain embodiments, the proximal umbilical cordis clamped, preferably within 4-5 cm (centimeter) of the insertion intothe placental disc prior to cord blood recovery. In other embodiments,the proximal umbilical cord is clamped after cord blood recovery butprior to further processing of the placenta.

Typically, a placenta is transported from the delivery or birthing roomto another location, e.g., a laboratory, for recovery of cord blood andcollection of stem cells by, e.g., perfusion or tissue dissociation. Inone embodiment, the placenta is preferably transported in a sterile,thermally insulated transport device (maintaining the temperature of theplacenta between 20-28° C.), for example, by placing the placenta, withclamped proximal umbilical cord, in a sterile plastic bag, which is thenplaced in an insulated container. In another embodiment, the placenta istransported in a cord blood collection kit substantially as described inpending U.S. patent application Ser. No. 11/230,760, filed Sep. 19,2005, which is incorporated herein by reference in its entirety.Preferably, the placenta is delivered to the laboratory four totwenty-four hours following delivery.

The placenta, prior to stem cell collection, can be stored under sterileconditions and at either room temperature or at a temperature of 5° C.to 25° C. (centigrade). The placenta may be stored for a period oflonger than forty eight hours, and or for a period of, e.g., four totwenty-four hours prior to perfusing the placenta to remove any residualcord blood. The placenta is preferably stored in an anticoagulantsolution at a temperature of 5° C. to 25° C. (centigrade). Suitableanticoagulant solutions are well known in the art. For example, asolution of heparin or warfarin sodium can be used. In a preferredembodiment, the anticoagulant solution comprises a solution of heparin(1% w/w in 1:1000 solution). The exsanguinated placenta is preferablystored for no more than 36 hours before placental stem cells arecollected.

While the placenta is awaiting stem cell collection, the placenta can becontacted with a solution comprising one or more oxygen-carryingperfluorocarbons (e.g., delivery room) to the site of stem cellcollection for at least part of the time between placental collectionand stem cell collection. Preferably, the placenta is kept in contactwith the oxygen-carrying perfluorocarbon for a majority of the timebetween placental collection and stem cell collection. The placenta canbe contacted with a composition comprising one or more oxygen-carryingperfluorocarbons and one or more organ preservation media (e.g., UWsolution; see Section 5.3, above) for at least part of the time betweenplacental collection and stem cell collection.

5.5 Placental Stem Cells

Placental stem cells that can be obtained in accordance with the methodsof the invention include pluripotent and multipotent cells, stem cellshaving one or more characteristics of embryonic stem cells, andcommitted progenitor cells.

Cord blood and placental blood contain predominantly CD34⁺CD38⁺hematopoietic progenitor cells. Within the first twenty-four hours afterexsanguination and removal of substantially all placental and umbilicalcord blood, high numbers of CD34⁺CD38-hematopoietic progenitor cells,relative to the number that may be found in cord blood or bone marrow,can be isolated from the placenta, along with high numbers of CD34⁻CD38⁺hematopoietic progenitor cells. After about twenty-four hours of cultureor maintenance in perfusion solution, high numbers of CD34⁻CD38⁻ cells,relative to the amount that can be obtained from, e.g., cord blood orbone marrow, can be isolated from the placenta along with theaforementioned cells.

In a preferred embodiment, placental stem cells obtained by the methodsof the invention are viable, quiescent, multipotent or pluripotent stemcells

At least one class of human placental stem cells is developmentallynaive. For example, such stem cells are SSEA3⁻ (stage-specific embryonicantigen 3), SSEA4⁻, OCT-4⁺ (a stem cell transcription factor) and ABC-p⁺(ATP-binding cassette (ABC) transporter protein), markers that areindicative of developmental naiveté. In a specific embodiment, the stemcells are non-embryonic and SSEA3⁻SSEA4⁻OCT-4⁺ ABC-p⁺. In anotherembodiment, the human placental stem cells do not express MHC Class 2antigens. In another embodiment, such stem cells are able todifferentiate into endodermal, ectodermal, or mesodermal cells.

In one embodiment, placental stem cells obtained by the methods of theinvention can be identified by the presence of the markers OCT-4 andABC-p. Further, the invention encompasses the collection of placentalstem cells displaying the markers CD10, CD29, CD44, CD54, CD90, CD105(SH2), CD73 (SH3, SH4), OCT-4, and ABC-p, or lacking or not displayingthe markers CD34, CD38, CD45, SSEA3, or SSEA4. In a specific embodiment,the placental stem cells are CD10⁺, CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45⁻,CD54⁺, CD73⁺, CD90⁺, CD105⁺, SSEA3⁻, SSEA4⁻, OCT-4⁺, and ABC-p⁺. Inanother specific embodiment, the placental stem cells are CD200⁺ andHLA-G⁺. In another specific embodiment, the placental stem cells areCD73⁺, CD105⁺ and CD200⁺. In another specific embodiment, the placentalstem cells are CD200⁺ and OCT-4⁺. In another specific embodiment, theplacental stem cells are CD73⁺ and CD105⁺, and a population of isolatedplacental cells comprising the placental stem cells forms embryoidbodies under conditions that allow the formation of embryoid bodies. Inanother specific embodiment, the placental stem cells are CD73⁺, CD105⁺and HLA-G⁺. In another specific embodiment, the placental stem cells areOCT-4⁺ and form embryoid bodies under conditions that allow theformation of embryoid bodies.

Such cell surface markers are routinely detected according to methodswell known in the art, e.g. by flow cytometry, followed by washing andstaining with an anti-cell surface marker antibody. For example, todetermine the presence of CD34 or CD38, cells may be washed in PBS andthen double-stained with anti-CD34 phycoerythrin and anti-CD38fluorescein isothiocyanate (Becton Dickinson, Mountain View, Calif.).

The placental stem cells that can be collected by the methods andcompositions of the invention can be used for a wide variety oftherapeutic protocols in which a tissue or organ of the body isaugmented, repaired or replaced by the engraftment, transplantation orinfusion of a desired cell population, such as a stem cell or progenitorcell population. The placental stem cells of the invention can be usedto replace or augment existing tissues, to introduce new or alteredtissues, or to join together biological tissues or structures. Theplacental stem cells of the invention can also be substituted forembryonic stem cells in therapeutic protocols in which embryonic stemcells would be typically be used.

6. EXAMPLES 6.1 Example 1 Stem Cell Collection Compositions

The following Example describes formulations of a stem cell collectioncomposition. Other embodiments will be apparent to one of skill in theart.

6.1.1 Compositions Comprising an Apoptosis Inhibitor and a Protease

A perfusion solution is made comprising 0.9% NaCl, caspase inhibitorAc-Val-Ala-Asp-CHO at a final concentration of 50-100 μM, andtrypsin-EDTA at a final concentration of about 0.25%.

A perfusion solution is made comprising the modified Krebs solutionshown in Example 1, caspase inhibitor Ac-Val-Ala-Asp-CHO at a finalconcentration of 50-100 μM, and trypsin-EDTA at a final concentration ofabout 0.25%.

A perfusion solution is made comprising the DMEM-based solution shown inExample 1, caspase inhibitor Ac-Val-Ala-Asp-CHO at a final concentrationof 50-100 μM, and trypsin-EDTA at a final concentration of about 0.25%.

6.1.2 Compositions Comprising a Combination of Apoptosis Inhibitors anda Protease

A perfusion solution is made comprising 0.9% NaCl, caspase inhibitorAc-Val-Ala-Asp-CHO at a final concentration of 50-100 μM, trypsin-EDTAat a final concentration of about 0.25%, and a JNK inhibitor having oneof the following structures:

wherein the JNK inhibitor is present at a concentration of about 0.5 μMto about 10 μM.

A perfusion solution is made comprising the DMEM-based solution shown inExample 1, caspase inhibitor Ac-Val-Ala-Asp-CHO at a final concentrationof 50-100 μM, a JNK inhibitor as shown above in this Example, andtrypsin-EDTA at a final concentration of about 0.25%.

A perfusion solution is made comprising the modified Krebs solutionshown in Example 1, caspase inhibitor Ac-Val-Ala-Asp-CHO at a finalconcentration of 50-100 μM, a JNK inhibitor as shown above in thisExample, and trypsin-EDTA at a final concentration of about 0.25%.

6.1.3 Compositions Comprising a JNK Inhibitor and a Combination ofProteases

A perfusion solution is made comprising 0.9% NaCl; a cocktail ofproteases comprising elastase at a final concentration of about 1.0mg/mL, collagenase I at a final concentration of about 1.0 mg/mL,collagenase IV at a final concentration of about 0.5 mg/mL, and dispaseat a final concentration of about 0.1 mg/ml; and a JNK inhibitor havingone of the following structures:

wherein the JNK inhibitor is present at a concentration of about 0.5 μMto about 10 μM.

A perfusion solution is made comprising the DMEM-based solution shown inExample 1; a cocktail of proteases comprising elastase at a finalconcentration of about 1.0 mg/mL, collagenase I at a final concentrationof about 1.0 mg/mL, collagenase IV at a final concentration of about 0.5mg/mL, and dispase at a final concentration of about 0.1 mg/mL; and aJNK inhibitor as shown above in this Example.

A perfusion solution is made comprising the modified Krebs solutionshown in Example 1; a cocktail of proteases comprising elastase at afinal concentration of about 1.0 mg/mL, collagenase I at a finalconcentration of about 1.0 mg/mL, collagenase IV at a finalconcentration of about 0.5 mg/mL, and dispase at a final concentrationof about 0.1 mg/mL; and a JNK inhibitor as shown above in this Example.

6.1.4 Compositions Comprising a Combination of JNK Inhibitors and aCombination of Enzymes

A perfusion solution is made comprising 0.9% NaCl; a cocktail ofproteases comprising elastase at a final concentration of about 1.0mg/mL, collagenase I at a final concentration of about 1.0 mg/mL,collagenase IV at a final concentration of about 0.5 mg/mL, dispase at afinal concentration of about 0.1 mg/mL, and hyaluronidase at aconcentration of about 0.1 to about 10.0 mg/mL; and two or more JNKinhibitors having one or more of the of the following structures:

wherein the JNK inhibitor is present at a concentration of about 0.5 μMto about 10 μM.

A perfusion solution is made comprising the DMEM-based solution shown inExample 1; a cocktail of proteases comprising elastase at a finalconcentration of about 1.0 mg/mL, collagenase I at a final concentrationof about 1.0 mg/mL, collagenase IV at a final concentration of about 0.5mg/mL, dispase at a final concentration of about 0.1 mg/mL, andhyaluronidase at a concentration of about 0.1 to about 10.0 mg/mL; andtwo or more of the JNK inhibitors as shown above in this Example.

A perfusion solution is made comprising the modified Krebs solutionshown in Example 1; a cocktail of proteases comprising elastase at afinal concentration of about 1.0 mg/mL, collagenase I at a finalconcentration of about 1.0 mg/mL, collagenase IV at a finalconcentration of about 0.5 mg/mL, dispase at a final concentration ofabout 0.1 mg/mL, and hyaluronidase at a concentration of about 0.1 toabout 10.0 mg/mL; and two or more of the JNK inhibitors as shown abovein this Example.

6.1.5 Compositions Comprising JNK Inhibitor, Protease and Vasodilator

A perfusion solution is made comprising 0.9% NaCl; trypsin-EDTA at afinal concentration of about 0.25%; magnesium sulfate as a vasodilatorat a concentration of about 0.15 mM to about 6 mM; and a JNK inhibitorhaving one of the following structures:

wherein the JNK inhibitor is present at a concentration of about 0.5 μMto 10 μM.

A perfusion solution is made comprising the DMEM-based solution shown inExample 1; trypsin-EDTA at a final concentration of about 0.25%;magnesium sulfate as a vasodilator at a concentration of about 0.15 mMto about 6 mM; and a JNK inhibitor as shown above in this Example.

A perfusion solution is made comprising the modified Krebs solutionshown in Example 1; trypsin-EDTA at a final concentration of about0.25%; magnesium sulfate as a vasodilator at a concentration of about0.15 mM to about 6 mM; and a JNK inhibitor as shown above in thisExample.

6.1.6 Compositions Comprising JNK Inhibitor, Protease and Anti-NecrosisAgent

A perfusion solution is made comprising 0.9% NaCl; trypsin-EDTA at afinal concentration of about 0.25%;2-(1H-Indol-3-yl)-3-pentylamino-maleimide at a concentration of about0.5 μM to about 100.0 μM; and a JNK inhibitor having one of thefollowing structures:

wherein the JNK inhibitor is present at a concentration of about 0.5 μMto about 10.0 μM.

A perfusion solution is made comprising the DMEM-based solution shown inExample 1; trypsin-EDTA at a final concentration of 0.25%;2-(1H-Indol-3-yl)-3-pentylamino-maleimide at a concentration of 0.5 μMto about 100.0 μM; and a JNK inhibitor as shown above in this Example.

A perfusion solution is made comprising the modified Krebs solutionshown in Example 1; trypsin-EDTA at a final concentration of 0.25%;2-(1H-Indol-3-yl)-3-pentylamino-maleimide at a concentration of 0.5 μMto about 100.0 μM; and a JNK inhibitor as shown above in this Example.

6.1.7 Compositions Comprising JNK Inhibitor, Protease andOxygen-Carrying Perfluorocarbon

A perfusion solution is made comprising 0.9% NaCl; trypsin-EDTA at afinal concentration of 0.25%; and a JNK inhibitor having one of thefollowing structures:

wherein the JNK inhibitor is present at a concentration of about 0.5 μMto about 10.0 μM. The solution also comprises perfluorooctylbromide at50%-100% weight to volume, perfluorodecylbromide at about 10% of thevolume of perfluorooctylbromide and lecithin as an emulsifier at about4% weight to volume. The solution is emulsified prior to use.

A perfusion solution is made as above, but comprising the DMEM-basedsolution shown in Example 1 in place of 0.9% NaCl. A perfusion solutionis made as above, but comprising the modified Krebs solution shown inExample 1 in place of 0.9% NaCl.

6.2 Example 2 Placental Stem Cell Culture

Placental stem cells are obtained from a post-partum mammalian placentaeither by perfusion or by physical disruption, e.g., enzymaticdigestion. The cells are cultured in a culture medium comprising 60%DMEM-LG (Gibco), 40% MCDB-201(Sigma), 2% fetal calf serum (FCS) (HycloneLaboratories), 1× insulin-transferrin-selenium (ITS), 1×lenolenic-acid-bovine-serum-albumin (LA-BSA), 10⁻⁹ M dexamethasone(Sigma), 10⁻⁴M ascorbic acid 2-phosphate (Sigma), epidermal growthfactor (EGF) 10 ng/ml (R&D Systems), platelet derived-growth factor(PDGF-BB) 10 ng/ml (R&D Systems), and 100U penicillin/1000Ustreptomycin.

The culture flask in which the cells are cultured is prepared asfollows. T75 flasks are coated with fibronectin (FN), by adding 5 ml PBScontaining 5 ng/ml human FN (Sigma F0895) to the flask. The flasks withFN solution are left at 37° C. for 30 min. The FN solution is thenremoved prior to cell culture. There is no need to dry the flasksfollowing treatment. Alternatively, the flasks are left in contact withthe FN solution at 4° C. overnight or longer; prior to culture, theflasks are warmed and the FN solution is removed.

Placental Stem Cells Isolated By Perfusion

Cultures of placental stem cells from placental perfusate areestablished as follows. Cells from a Ficoll gradient are seeded inFN-coated T75 flasks, prepared as above, at 50-100×10⁶ cells/flask in 15ml culture medium. Typically, 5 to 10 flasks are seeded. The flasks areincubated at 37° C. for 12-18 hrs to allow the attachment of adherentcells. 10 ml of warm PBS is added to each flask to remove cells insuspension, and mixed gently. 15 mL of the medium is then removed andreplaced with 15 ml fresh culture medium. All medium is changed 3-4 daysafter the start of culture. Subsequent culture medium changes areperformed, during which 50% or 7.5 ml of the medium is removed.

Starting at about day 12, the culture is checked under a microscope toexamine the growth of the adherent cell colonies. When cell culturesbecome approximately 80% confluent, typically between day 13 to day 18after the start of culture, adherent cells are harvested by trypsindigestion. Cells harvested from these primary cultures are designatedpassage 0 (zero).

Placental Stem Cells Isolated By Enzymatic Digestion

Placental stem cell cultures are established from digested placentaltissue as follows. The perfused placenta is placed on a sterile papersheet with the maternal side up. Approximately 0.5 cm of the surfacelayer on maternal side of placenta is scraped off with a blade, and theblade is used to remove a placental tissue block measuring approximately1×2×1 cm. This placenta tissue is then minced into approximately 1 mm³pieces. These pieces are collected into a 50 ml Falcon tube and digestedwith collagenase IA (2 mg/ml, Sigma) for 30 minutes, followed bytrypsin-EDTA (0.25%, GIBCO BRL) for 10 minutes, at 37° C. in water bath.The resulting solution is centrifuged at 400 g for 10 minutes at roomtemperature, and the digestion solution is removed. The pellet isresuspended to approximately 10 volumes with PBS (for example, a 5 mlpellet is resuspended with 45 ml PBS), and the tubes are centrifuged at400 g for 10 minutes at room temperature. The tissue/cell pellet isresuspended in about 130 ml culture medium, and the cells are seeded at13 ml per fibronectin-coated T-75 flask. Cells are incubated at 37° C.with a humidified atmosphere with 5% CO₂. Placental Stem Cells areoptionally cryopreserved at this stage.

Subculturing and Expansion of Placental Stem Cells

Cryopreserved cells are quickly thawed in a 37° C. water bath. Placentalstem cells are immediately removed from the cryovial with 10 ml warmmedium and transferred to a 15 ml sterile tube. The cells arecentrifuged at about 400×g for 5 minutes at room temperature. The cellsare gently resuspended in 10 ml of warm culture medium by pipetting, andviable cell counts are determined by Trypan blue exclusion. Cells arethen seeded at about 6000-7000 cells per cm² onto FN-coated flasks,prepared as above (approximately 5×10⁵ cells per T-75 flask). The cellsare incubated at 37° C., 5% CO₂ and 90% humidity. When the cells reached75-85% confluency, all of the spent media is aseptically removed fromthe flasks and discarded. 3 ml of trypsin/EDTA solution, 0.25%, is addedto cover the cell layer, and the cells are incubated at 37° C., 5% CO²and 90% humidity for 5 minutes. The flask is tapped once or twice toexpedite cell detachment. Once >95% of the cells are rounded anddetached, 7 ml of warm culture medium is added to each T-75 flask, andthe solution is dispersed by pipetting over the cell layer surfaceseveral times.

After counting the cells and determining viability as above, the cellsare centrifuged at 1000 RPM for 5 minutes at room temperature. Cells arepassaged by gently resuspending the cell pellet from one T-75 flask withculture medium, and evenly plating the cells onto two FN-coated T-75flasks.

Using the above methods, populations of adherent placental stem cellsare identified that express markers CD105, CD117, CD33, CD73, CD29,CD44, CD10, CD90 and CD133. This population of cells did not expressCD34 or CD45. Some, but not all cultures of these placental stem cellsexpressed HLA-ABC and/or HLA-DR.

6.3 Example 3 Differentiation of Placental Stem Cells

6.3.1 Induction of Differentiation into Neurons

Neuronal differentiation of placental stem cells can be accomplished,for example, as follows:

-   -   1. Placental stem cells are grown for 24 hr in preinduction        medium consisting of DMEM/20% FBS and 1 mM beta-mercaptoethanol.    -   2. The preinduction medium is removed and cells are washed with        PBS.    -   3. Neuronal induction medium consisting of DMEM and 1-10 mM        betamercaptoethanol is added to the cells. Alternatively,        induction media consisting of DMEM/2% DMSO/200 μM butylated        hydroxyanisole may be used.    -   4. In certain embodiments, morphologic and molecular changes may        occur as early as 60 minutes after exposure to serum-free media        and betamercaptoethanol. RT/PCR may be used to assess the        expression of e.g., nerve growth factor receptor and        neurofilament heavy chain genes.

6.3.2 Induction of Differentiation into Adipocytes

Adipogenic differentiation of placental stem cells can be accomplished,for example, as follows:

-   -   1. Placental stem cells are grown in MSCGM (Cambrex) or DMEM        supplemented with 15% cord blood serum.    -   2. Three cycles of induction/maintenance are used. Each cycle        consists of feeding the placental stem cells with Adipogenesis        Induction Medium (Cambrex) and culturing the cells for 3 days        (at 37° C., 5% CO₂), followed by 1-3 days of culture in        Adipogenesis Maintenance Medium (Cambrex). An alternate        induction medium that can be used contains 1 μM dexamethasone,        0.2 mM indomethacin, 0.01 mg/ml insulin, 0.5 mM IBMX, DMEM-high        glucose, FBS, and antibiotics.    -   3. After 3 complete cycles of induction/maintenance, the cells        are cultured for an additional 7 days in adipogenesis        maintenance medium, replacing the medium every 2-3 days.    -   4. A hallmark of adipogenesis is the development of multiple        intracytoplasmic lipid vesicles that can be easily observed        using the lipophilic stain oil red O. Expression of lipase        and/or fatty acid binding protein genes is confirmed by RT/PCR        in placental stem cells that have begun to differentiate into        adipocytes.

6.3.3 Induction of Differentiation into Chondrocytes

Chondrogenic differentiation of placental stem cells can beaccomplished, for example, as follows:

-   -   1. Placental stem cells are maintained in MSCGM (Cambrex) or        DMEM supplemented with 15% cord blood serum.    -   2. Placental stem cells are aliquoted into a sterile        polypropylene tube. The cells are centrifuged (150×g for 5        minutes), and washed twice in Incomplete Chondrogenesis Medium        (Cambrex).    -   3. After the last wash, the cells are resuspended in Complete        Chondrogenesis Medium (Cambrex) containing 0.01 μg/ml TGF-beta-3        at a concentration of 5×10(5) cells/ml.    -   4. 0.5 ml of cells is aliquoted into a 15 ml polypropylene        culture tube. The cells are pelleted at 150×g for 5 minutes. The        pellet is left intact in the medium.    -   5. Loosely capped tubes are incubated at 37° C., 5% CO² for 24        hours.    -   6. The cell pellets are fed every 2-3 days with freshly prepared        complete chondrogenesis medium.    -   7. Pellets are maintained suspended in medium by daily agitation        using a low speed vortex.    -   8. Chondrogenic cell pellets are harvested after 14-28 days in        culture.    -   9. Chondrogenesis is characterized by e.g., observation of        production of esoinophilic ground substance, assessing cell        morphology, an/or RT/PCR confirmation of collagen 2 and collagen        9 gene expression.

6.3.4 Induction of Differentiation into Osteocytes

Osteogenic differentiation can be accomplished, for example, as follows:

-   -   1. Adherent cultures of placental stem cells are cultured in        MSCGM (Cambrex) or DMEM supplemented with 15% cord blood serum.    -   2. Cultures are cultured for 24 hours in tissue culture flasks.    -   3. Osteogenic differentiation is induced by replacing MSCGM with        Osteogenic Induction Medium (Cambrex) containing 0.1 μM        dexamethasone, 0.05 mM ascorbic acid-2-phosphate, 10 mM beta        glycerophosphate.    -   4. Cells are fed every 3-4 days for 2-3 weeks with Osteogenic        Induction Medium.    -   5. Differentiation is assayed using a calcium-specific stain and        RT/PCR for alkaline phosphatase and osteopontin gene expression.

6.3.5 Induction of Differentiation into Pancreatic Cells

Pancreatic differentiation can be accomplished, for example, as follows:

-   -   1. Placental stem cells are cultured in DMEM/20% CBS,        supplemented with basic fibroblast growth factor (10 ng/ml),        nicotinamide (10 mM), B27 (1×), N2 (1×), and transforming growth        factor beta-1, 2 ng/ml. KnockOut Serum Replacement may be used        in lieu of CBS.    -   2. Conditioned media from nestin-positive neuronal cell cultures        is added to media at a 50/50 concentration.    -   3. Cells are cultured for 14-28 days, refeeding every 3-4 days.    -   4. Differentiation is characterized by assaying for insulin        protein or insulin gene expression by RT/PCR.

6.3.6 Induction of Differentiation into Cardiac Cells

Myogenic (cardiogenic) differentiation can be accomplished, for example,as follows:

-   -   1. Placental stem cells are cultured in DMEM/20% CBS,        supplemented with retinoic acid, 1 μM; basic fibroblast growth        factor, 10 ng/ml; and transforming growth factor beta-1, 2        ng/ml; and epidermal growth factor, 100 ng/ml. KnockOut Serum        Replacement (Invitrogen, Carlsbad, Calif.) may be used in lieu        of CBS.    -   2. Alternatively, placental stem cells are cultured in DMEM/20%        CBS supplemented with 50 ng/ml Cardiotropin-1 for 24 hours.    -   3. Alternatively, placental stem cells are maintained in        protein-free media for 5-7 days, then stimulated with human        myocardium extract (escalating dose analysis). Myocardium        extract is produced by homogenizing 1 gm human myocardium in 1%        HEPES buffer supplemented with 1% cord blood serum. The        suspension is incubated for 60 minutes, then centrifuged and the        supernatant collected.    -   4. Cells are cultured for 10-14 days, refeeding every 3-4 days.    -   5. Differentiation is confirmed by demonstration of cardiac        actin gene expression by RT/PCR.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention.

1. A composition comprising, in a physiologically-acceptable solution,an inhibitor of apoptosis and a protease, wherein said composition, whencontacted with population of stem cells, reduces or prevents apoptosisin said population of stem cells as compared to a population of stemcells not contacted with the composition, and wherein said compositionis not a naturally-occurring composition.
 2. The composition of claim 1,wherein said inhibitor of apoptosis is a caspase inhibitor.
 3. Thecomposition of claim 1, wherein said inhibitor of apoptosis is a JNKinhibitor.
 4. The composition of claim 1, wherein said JNK inhibitordoes not modulate differentiation or proliferation of said stem cells.5. The composition of claim 1, wherein said protease is present in anamount sufficient to detectably dissociate the cells of a tissue fromwhich said stem cells may be derived.
 6. The composition of claim 1,additionally comprising an inhibitor of necrosis.
 7. The composition ofclaim 6, wherein said inhibitor of necrosis is2-(1H-Indol-3-yl)-3-pentylamino-maleimide.
 8. The composition of claim1, additionally comprising an oxygen-carrying perfluorocarbon.
 9. Thecomposition of claim 1 wherein said physiologically-acceptable solutionis a saline solution or culture medium.
 10. The composition of claim 9wherein said saline solution is 0.9% NaCl solution or phosphate bufferedsaline.
 11. The composition of claim 1, wherein said protease is amatrix metalloprotease or a neutral protease.
 12. The composition ofclaim 11, wherein said matrix metalloprotease is collagenase.
 13. Thecomposition of claim 11, wherein said neutral protease is thermolysin ordispase.
 14. The composition of claim 11 additionally comprising amucolytic enzyme.
 15. The composition of claim 14, wherein saidmucolytic enzyme is hyaluronidase.
 16. The composition of claim 1,additionally comprising hydroxyethyl starch, lactobionic acid andraffinose.
 17. The composition of claim 1, additionally comprising UWsolution.
 18. The composition of claim 1, wherein said JNK inhibitor isan indazole.
 19. The composition of claim 1, wherein said JNK inhibitorhas the structure

wherein: A is a direct bond, —(CH₂)_(a)—, —(CH₂)_(b)CH═CH(CH₂)_(c)—, or—(CH₂)_(b)C≡C(CH₂)_(n)—; R₁ is aryl, heteroaryl or heterocycle fused tophenyl, each being optionally substituted with one to four substituentsindependently selected from R₃; R₂ is —R₃, —R₄, —(CH₂)_(b)C(═O)R₅,—(CH₂)_(b)C(═O)OR⁵, —(CH₂)_(b)C(═O)NR₅R₆,—(CH₂)_(b)C(═O)NR₅(CH₂)_(c)C(═O)R₆, —(CH₂)_(b)NR₅C(═O)R₆,—(CH₂)_(b)NR₅C(═O)NR₆R₇, —(CH₂)_(b)NR₅R₆, —(CH₂)_(b)OR₅,—(CH₂)_(b)SO_(d)R₅ or —(CH₂)_(b)SO₂NR₅R₆; a is 1, 2, 3, 4, 5 or 6; b andc are the same or different and at each occurrence independentlyselected from 0, 1, 2, 3 or 4; d is at each occurrence 0, 1 or 2; R₃ isat each occurrence independently halogen, hydroxy, carboxy, alkyl,alkoxy, haloalkyl, acyloxy, thioalkyl, sulfinylalkyl, sulfonylalkyl,hydroxyalkyl, aryl, arylalkyl, heterocycle, heterocycloalkyl, —C(═O)OR₈,—OC(═O)R₈, —C(═O)NR₈R₉, —C(═O)NR₈OR₉, —SO₂NR₈R₉, —NR₈SO₂R₉, —CN, —NO₂,—NR₈R₉, —NR₉C(═O)R₉, —NR₈C(═O)(CH₂)_(b)OR₉, —NR₉C(═O)(CH₂)_(b)R₉,—O(CH₂)_(b)NR₉R₉, or heterocycle fused to phenyl; R₄ is alkyl, aryl,arylalkyl, heterocycle or heterocycloalkyl, each being optionallysubstituted with one to four substituents independently selected fromR₃, or R₄ is halogen or hydroxy; R₅, R₆ and R₇ are the same or differentand at each occurrence independently hydrogen, alkyl, aryl, arylalkyl,heterocycle or heterocycloalkyl, wherein each of R₅, R₆ and R₇ areoptionally substituted with one to four substituents independentlyselected from R₃; and R₈ and R₉ are the same or different and at eachoccurrence independently hydrogen, alkyl, aryl, arylalkyl, heterocycle,or heterocycloalkyl, or R₈ and R₉ taken together with the atom or atomsto which they are bonded form a heterocycle, wherein each of R₅, R₉, andR⁸ and R₉ taken together to form a heterocycle are optionallysubstituted with one to four substituents independently selected fromR₃; or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.20. The composition of claim 1, wherein said JNK inhibitor has thestructure

wherein: R₁ is aryl or heteroaryl optionally substituted with one tofour substituents independently selected from R₇; R₂ is hydrogen; R₃ ishydrogen or lower alkyl; R₄ represents one to four optionalsubstituents, wherein each substituent is the same or different andindependently selected from halogen, hydroxy, lower alkyl and loweralkoxy; R₅ and R₆ are the same or different and independently —R₈,—(CH₂)_(a)C(═O)R₉, —(CH₂)_(a)C(═O)OR₉, (CH₂)_(a)C(═O)NR₉R₁₀,(CH₂)_(a)C(═O)NR₉(CH₂)_(b)C(═O)R₁₀, —(CH₂)_(a)NR₉C(═O)R₁₀,—(CH₂)_(a)NR₁₁C(═O)NR₉R₁₀, —(CH₂)_(a)NR₉R₁₀, —(CH₂)_(a)OR₉,—(CH₂)_(a)SO_(c)R₉ or —(CH₂)_(a)SO₂NR₉R₁₀; or R₅ and R₆ taken togetherwith the nitrogen atom to which they are attached to form a heterocycleor substituted heterocycle; R₇ is at each occurrence independentlyhalogen, hydroxy, cyano, nitro, carboxy, alkyl, alkoxy, haloalkyl,acyloxy, thioalkyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, aryl,arylalkyl, heterocycle, substituted heterocycle, heterocycloalkyl,—C(═O)OR₈, —OC(═O)R₈, —C(═O)NR₈R₉, —C(═O)NR₈OR₉, —SO_(c)R₈,—SO_(c)NR₈R₉, —NR₈SO_(c)R₉, —NR₈R₉, —NR₈C(═O)R₉, —NR₉C(═O)(CH₂)_(b)OR₉,—NR₈C(═O)(CH₂)_(b)R₉, —O(CH₂)_(b)NR₈R₉, or heterocycle fused to phenyl;R₈, R₉, R₁₀ and R₁₁ are the same or different and at each occurrenceindependently hydrogen, alkyl, aryl, arylalkyl, heterocycle,heterocycloalkyl; or R₈ and R₉ taken together with the atom or atoms towhich they are attached to form a heterocycle; a and b are the same ordifferent and at each occurrence independently selected from 0, 1, 2, 3or 4; and c is at each occurrence 0, 1 or 2; or a pharmaceuticallyacceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer,racemate, or mixture of stereoisomers thereof.
 21. The composition ofclaim 1, wherein said JNK inhibitor has the structure

wherein R₀ is —O—, —S—, —S(O)—, —S(O)₂—, NH or —CH₂—; the compound ofstructure (III) being: (i) unsubstituted, (ii) monosubstituted andhaving a first substituent, or (iii) disubstituted and having a firstsubstituent and a second substituent; the first or second substituent,when present, is at the 3, 4, 5, 7, 8, 9, or 10 position, wherein thefirst and second substituent, when present, are independently alkyl,hydroxy, halogen, nitro, trifluoromethyl, sulfonyl, carboxyl,alkoxycarbonyl, alkoxy, aryl, aryloxy, arylalkyloxy, arylalkyl,cycloalkylalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy,aminoalkoxy, mono-alkylaminoalkoxy, di-alkylaminoalkoxy, or a grouprepresented by structure (a), (b), (c), (d), (e), or (f):

wherein R₃ and R₄ are taken together and represent alkylidene or aheteroatom-containing cyclic alkylidene or R₃ and R₄ are independentlyhydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,aryloxyalkyl, alkoxyalkyl, aminoalkyl, mono-alkylaminoalkyl, ordi-alkylaminoalkyl; and R₅ is hydrogen, alkyl, cycloalkyl, aryl,arylalkyl, cycloalkylalkyl, alkoxy, alkoxyalkyl, alkoxycarbonylalkyl,amino, mono-alkylamino, di-alkylamino, arylamino, arylalkylamino,cycloalkylamino, cycloalkylalkylamino, aminoalkyl, mono-alkylaminoalkyl,or di-alkylaminoalkyl; or a pharmaceutically acceptable salt, hydrate,solvate, clathrate, enantiomer, diastereomer, racemate, or mixture ofstereoisomers thereof
 22. The composition of claim 1, additionallycomprising an immunomodulatory compound.
 23. The composition of claim22, wherein said immunomodulatory compound is3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione;3-(4′aminoisolindoline-1′-onw)-1-piperidine-2,6-dione;4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; orα-(3-aminophthalimido) glutarimide.
 24. The composition of claim 22,wherein said immunomodulatory compound is a compound having thestructure

wherein one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl, or a pharmaceutically acceptable salt,hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, ormixture of stereoisomers thereof
 25. The composition of claim 22,wherein said immunomodulatory compound is a compound having thestructure

wherein one of X and Y is C═O and the other is CH₂ or C═O; R¹ is H,(C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl,aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴,(C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³,C(S)NHR³, C(O)NR³R³, C(S)NR³R³ or (C₁-C₈)alkyl-O(CO)R⁵; R² is H, F,benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl; R³ and R^(3′)are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-C₂-C₅)heteroaryl, (C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵,(C₁-C₈)alkyl-C(O)OR⁵, (C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵; R⁴ is(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵, benzyl,aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, or(C₀-C₄)alkyl-C₂-C₅)heteroaryl; R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, or (C₂-C₅)heteroaryl; each occurrence ofR⁶ is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,benzyl, aryl, (C₂-C₅)heteroaryl, or (C₀-C₈)alkyl-C(O)O—R⁵ or the R⁶groups can join to form a heterocycloalkyl group; n is 0 or 1; and *represents a chiral-carbon center; or a pharmaceutically acceptablesalt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate,or mixture of stereoisomers thereof
 26. The composition of claim 22,wherein said immunomodulatory compound is a compound having thestructure

wherein: one of X and Y is C═O and the other is CH₂ or C═O; R is H orCH₂OCOR′; (i) each of R¹, R², R³, or R⁴, independently of the others, ishalo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or(ii) one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹,R², R³, or R⁴ are hydrogen; R⁵ is hydrogen or alkyl of 1 to 8 carbons R⁶hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro; R′ isR⁷—CHR¹⁰—N(R⁸R⁹); R⁷ is m-phenylene or p-phenylene or —(C_(n)H_(2n))— inwhich n has a value of 0 to 4; each of R⁸ and R⁹ taken independently ofthe other is hydrogen or alkyl of 1 to 8 carbon atoms, or R⁸ and R⁹taken together are tetramethylene, pentamethylene, hexamethylene, or—CH₂CH₂X₁CH₂CH₂— in which X₁ is —O—, —S—, or —NH—; R¹⁰ is hydrogen,alkyl of to 8 carbon atoms, or phenyl; and * represents a chiral-carboncenter; or a pharmaceutically acceptable salt, hydrate, solvate,clathrate, enantiomer, diastereomer, racemate, or mixture ofstereoisomers thereof.
 27. The composition of claim 1, wherein saidperfusion solution additionally comprises a vasodilator.
 28. Thecomposition of claim 27, wherein said vasodilator is an antihypertensivedrug.
 29. The composition of claim 27, wherein said vasodilatoractivates guanylyl cyclase, ADP-ribosyl transferase or cyclooxygenase,or inhibits lipoxygenase.
 30. The composition of claim 27, wherein saidvasodilator is atrial natriuretic peptide (ANP), adrenocorticotropin,corticotropin-releasing hormone, sodium nitroprusside, hydralazine,adenosine triphosphate, adenosine, indomethacin or magnesium sulfate.31. The composition of claim 30, wherein said hydralazine is present ina concentration of from about 0.1 mM to about 10 mM.
 32. The compositionof claim 30, wherein said adenosine is present at a concentration ofabout 0.001 mM to about 10.0 mM.
 33. The composition of claim 30,wherein said adenosine triphosphate is present at a concentration ofabout 0.1 mM to about 1000 mM.
 34. The composition of claim 30, whereinsaid indomethacin is present at a concentration of about 1 mg/kg toabout 20 mg/kg, wherein “kg” is the weight of the placenta.
 35. Thecomposition of claim 30, wherein said magnesium sulfate is present at aconcentration of about 0.1 mM to about 20 mM.